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Purpose Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth. Methods MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry). Results GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization. Conclusion GNP-HCPe treatment displays in vitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy.

The role of CD19+CD24From Jan 2009 to Dec 2014, 117 lung Tx recipients were submitted to an immunological follow up I-FU(median: 108.7 months (6.7-310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD19+CD24Among all variables analyzed at multivariate analysis: chronic rejection (OR - 0.19, p = .039), use of Mycophenolate (OR - 0.38, p .001) and the presence of a concomitant pulmonary infection of S. aureus (OR 0.66, p = .002) and A. fumigatus (OR 0.50, p = .009) were significantly associated to B-reg cell. No significant correlation between CD19+CD24Our present data highlight, for the first time, that this cell subset might participate in long-term lung graft acceptance mechanisms.

Emanuela Cova,1 Laura Pandolfi,1 Miriam Colombo,2 Vanessa Frangipane,1 Simona Inghilleri,1 Monica Morosini,1 Simona Mrakic-Sposta,3 Sarah Moretti,3 Manuela Monti,4 Ymera Pignochino,5 Silvia Benvenuti,5 Davide Prosperi,2,6 Giulia Stella,1 Patrizia Morbini,7 Federica Meloni8 1Clinic of Lung Diseases, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 2Deparment of Biotechnology and Bioscience, University of Milano - Bicocca, Milan, Italy; 3National Council of Research, Institute of Bioimaging and Molecular Physiology, Segrate, Milan, Italy; 4Laboratory of Biotechnology, Research Center of Rigenerative Medicine, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 5Experimental Clinical Molecular Oncology, IRCCS Candiolo Cancer Institute-FPO, Candiolo, Turin, Italy; 6Laboratory of Nanomedicine, Clinical Institute of Maugeri, S.p.A., Pavia, Italy; 7Department of Molecular Medicine, Pathology Unit, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 8Department of Internal Medicine, Pneumology Unit, University of Pavia, Pavia,Italy Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth.Methods: MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry).Results: GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization.Conclusion: GNP-HCPe treatment displays invitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy. Keywords: gold nanoparticles, nanodrug delivery, mesothelioma, pemetrexed, intrapleural delivery

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis, mainly associated with work or environmental exposure to asbestos. MPM’s molecular profile is largerly unexplored and effective therapies are still lacking. MPM rarely harbours those somatic genetic lesions that usually characterize solid epithelial-derived tumors. On this basis, our study aims at investigating MPM epigenetic profile. Methods: We here assessed through immunohistochemistry, FISH and methylation specific PCR, the expression of 5-hydroxymethylcytosine (5- hmC) - an epigenetic marker and an important regulator of embryonic development and carcinogenesis - and the methylation status of the promoter of the MTAP gene - encoding for an enzyme involved in the rescue process of methionine and adenine - in two relevant series of FF-PE MPM samples derived from MPM thoracoscopic biopsies. Tissue sampling was performed at diagnosis. Results: Within the limitations of the study cohort, the 5-hmC immunophenotype was different among the histological MPM types analysed. In fact, 18% of the epithelial MPMs were negative, 47% weakly positive, and 35% of the cases showed an intense expression of 5-hmC. Sarcomatoid and biphasic MPMs showed intense 5-hmC expression pattern (positive and weakly positive in more than 80% of cases). Among MPM featuring epithelial lineage, none showed methylation of MTAP promoter. Conclusions: Mesothelial sarcomatoid tumors featured a methylation profile characterized by a permanent gene silencing. Epithelial MPM methylation profile was in-between that of sarcomatoid MPM and the one of epithelial-derived tumors. MTAP promoter methylation level cannot be considered a suitable biomarker of epithelial MPM arousal.

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis and continuously increasing incidence due to widespread exposure to asbestos. Novel approaches for MPM management could take advantage of the intrapleural administration of drugs. Our group has recently developed a novel nanoplatform using gold nanoparticles (GNPs) aimed at the local treatment of lung diseases [Cova E et al. 2015]. We already proved that engineered GNPs loaded with pemetrexed and specifically decorated with the anti-CD146 expressed by MPM cells, were highly effective in inhibiting cancer cells [Stella GM et al. Eur Respir J 2015 46: OA5003]. The MAP kinase pathway is known to regulate proliferation and survival of tumor cells, including MPM [Myioshy S et al. 2012]. Clinical improvement of selumetinib slowed down due to reported adverse events [Janne PA et al, 2013]. Here, we aimed at investigating the therapeutic efficacy of selumetinib on MPM. Therefore, we preliminary tested selumetinib on two different MPM cell lines, MSTO-211H and H2452, representative of biphasic and epithelioid subtypes respectively (Fig.1). We found that selumetinib was efficacy in inhibiting MSTO-211H line as evidenced by half maximal inhibitory concentration (IC50) of 1.59 uM after 96 h incubation. However, selumetinib was almost ineffective in inhibiting H2452 cells by using the same drug concentrations suggesting a resistance to MEK inhibitors, as already observed for melanoma cell lines (Emery et al., 2009). At least in selected cases, selumetinib might be an useful drug to be loaded by functionalized targeted GNPs

We demonstrated that targeted-gold-nanoparticles loaded with everolimus (GNP-HCe) were effective in vitro inhibiting mesenchymal cells isolated from bronchoalveolar lavage of BOS patients. Here, we investigated the effect of GNPs on inflammatory cells, given that a stimulating effect is strongly unwanted. Macrophages were obtained from BAL by adhesion procedure, lymphocytes and neutrophils from peripheral blood by Lympholyte. Cells were incubated with GNP-HC (targeted GNP without everolimus), GNP-HCe, everolimus (EV) and medium. IL-8 was assayed by ELISA, IFN-g, IL-17 and IL-10 by Elispot. Elastase release was evaluated by enzymatic test. Apoptosis, CD3+CD4+, CD3+CD8+ populations and CCR7 expression were analyzed by flow cytometry, cell viability by MTT test, and mitochondria membrane potential by JC-1 fluorescence. Reactive oxygen species (ROS) production was assayed by EPR spectroscopy. GNP-HCe did not stimulate IL-8 secretion by macrophages but reduced cell viability (p Our study confirm that GNP-HCe are able to exert its therapeutic action without activating immune effectors with positive implications on the translational potential of this therapeutic strategy.

Our work has the objective to develop and provide a new therapeutic approach to rare respiratory disease with poor prognosis. These diseases are united by the fact of being rare diseases, defined orphan, with a very poor prognosis and limited treatment options. Furthermore, they share the possibility to apply a local treatment with the advantage of decreased unwanted biodistribution and systemic toxicity. Bronchiolitis obliterans (BO) is a disease characterized by fibrotic obliteration of the small airways that occurs in response to inflammatory and immunological insults. Malignant pleural mesothelioma (MPM) is a rare malignant tumor that originates from the pleura, strongly associated with environmental exposure to asbestos. Our approach consists in the creation of nanocarriers (gold nanoparticles, GNPs), which can be loaded with specific anti-proliferative drugs, specifically targeted to the cells responsible of the two pathological processes (fibroblastoid-like mesenchymal cells in BO, malignant mesothelioma cells in MPM) and suitable for administration by local street (inhaled or intrapleural). First, we isolated, cultured and phenotyped primary cells from patients from BO and MPM. Once you have identified some targets (CD44 for the BO and CD146 for MPM) we designed a nanotool loaded with the specific drugs (everolimus or pemetrexed) and decorated with monoclonal antibody on the surface. We performed experiments in vitro on primary cultures of pathological cells and we demonstrated that these nanoparticles were able to penetrate specifically in target cells expressing the specific receptor and not in other types of normal cells tested, except for the alveolar macrophage which presented the tendency to absorb the nanoparticles, also the not functionalized ones. The anti-proliferative, pro-apoptotic and anti-inflammatory action of these nanoparticles was tested in vitro. We have also conducted experiments in animals to prove the lack of both pulmonary and extrapulmonary toxicity following administration of nanoparticles by inhalation.

The use of gold nanoparticles as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favours epithelial mesenchymal transition and activation of local mesenchymal cells leading to airway fibro-obliteration. In a previous work, we engineered gold nanoparticles loaded with the mTOR inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary mesenchymal cells isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these gold nanoparticles (GNP-HCe) were able to specifically inhibit primary mesenchymal cells without affecting bronchial epithelial cell. In the present work, we investigated the effect of the these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibro-genesis. In addition, we administered GNP-HCe by inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. Present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are nontoxic.

Background Systemic Sclerosis (SSc) is an autoimmune disorder frequently affected by an interstitial lung involvement (ILD) that significantly deteriorates long term outcomes. In previous experiments we proved that specifically engineered gold-nanoparticles (GNP) loaded with imatinib and targeted with an anti CD44 Ab (GNP-HCim) significantly inhibited proliferation and induced apoptosis of fibroblast-like cells derived from ILD-SSc patients [1]. In vitro, GNP-HCim showed higher efficacy compared to the drug alone. Objectives To demonstrate in vivo the efficacy of GNP-HCim in ameliorating bleomycin-induced lung fibrosis Methods Eight-week-old C57BL6 male mice (n=8/group) were assigned to either: (1) controls receiving intratracheal aereosolization of saline solution and unloaded functionalised GNP (GNP-HC); (2) mice treated with intratracheal instillation of bleomycin (50 uL) on day 0 and GNP-HC; (3) mice treated with bleomycin on day 0 plus GNP-HCim; (4) mice treated with bleomycin plus intraperitoneal (i.p.) Imatinib (50 mg/kg, once daily). GNP-HC or GNP-HCim were administered by intratracheal instillation on day 10–15–20–25 and 3 h before culling. All mice were sacrificed on day 28. Lung specimens were analysed by electron microscopy, immunohistochemistry and immunofluorescence (IF). Data were evaluated by 2 blind observers and analysed with GraphPrism software for statistics. Results The administration of imatinib i.p or via GNP-HC reduced pathologic changes of the lungs as evaluated by the Lung Injury score and the Ashcroft score (p Conclusions In the experimental model of bleomycin-induced lung fibrosis imatinib delivered to lungs through inhalation of anti CD44 targeted GNP was as effective as imatinib administered by i.p. route. These data favour the use of GNP in the development of new therapeutic approaches to SSc-ILD, which might be associated to a lower toxicity and side effects of systemic treatment. References Cova E, Ann rheum Dis 2016, 75(Suppl2): 61. Disclosure of Interest None declared

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit—in a similar fashion to cancer—targeted therapies for a “precision medicine” approach to IPF.

Background The role of CD19+CD24highCD38high B-regulatory cells in solid-organ Transplant (Tx) in acceptance are still scarce. In previous studies on kidney transplant recipients may suggest a protective role of this cell subtype in graft tolerance and the existence of a cross talk between B-and T-regulatory clones. In lung transplantation, the role of B-regulatory cells has never been investigated. In a murine tracheal transplantation model, this subset seems able to prevent tracheal obliteration when in combination with rapamycin. Aim of this study is to analyze peripheral CD19+CD24highCD38high B-reg cells counts in a cohort of lung recipients, their association with several clinical and pharmacological variables and their possible association with T regulatory cell. Methods From Jan 2009 to Dec 2014, 117 lung Tx recipients were submitted to an immunological follow up I-FU(median: 108.7 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD19+CD24highCD38high B-cells (globally 1106 determinations). We tested the association between B-reg and relevant variables by linear or regression models for repeated measures, adjusting for time from Tx. Results Among all variables analyzed at multivariate analysis: chronic rejection (OR − 0.19, p = .039), use of Mycophenolate (OR − 0.38, p No significant correlation between CD19+CD24highCD38high B-reg cells and T-reg cells counts was found in our cohort. Conclusions Our present data highlight, for the first time, that this cell subset might participate in long-term lung graft acceptance mechanisms.

The presence of activating mutations of the epidermal growth factor receptor (EGFR)-gene identifies a distinct and clinically relevant molecular subset of non-small-cell lung cancer. It is now well demonstrated that EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are superior to standard chemotherapy in this subset of tumors. Nevertheless, in many cases, responses are not durable and last for 6–12 months due to the occurrence of secondary or acquired resistance. Here we present three cases of EGFR-mutant lung adenocarcinomas (ADC), that showed an unexpected response to anti-EGFR small molecules. The first patient presented a continued 89 month-long response to erlotinib in a tumor recurred after surgery and conventional chemotherapy. In the other cases, subclinically persistent tumor in the lung tissue was documented histologically in lung resections performed after partial response to TKI treatment. The persistence of interstitial and endolymphatic tumor cells after TKI treatment might explain the common observation of tumor relapse after TKI discontinuation, and sustain the decision to continue treatment in responsive patients as in our first case.

Interstitial lung disease (SSc-ILD) significantly limits long term outcome in Systemic Sclerosis. Gold nanoparticles (GNPs) have a great potential for targeted drug-delivery as we previously showed using drug-loaded GNPs targeted (with anti CD44Ab) to mesenchymal cells obtained from patients with chronic lung rejection. Since we assessed that lung fibroblastoid-like cells (FLCs) isolated from ILD-SSc patients express high rate of CD44, we tested on these cells in vitro anti-CD44 functionalized gold nanoparticles loaded with imatinib (GNP-HCim). GNP-HCim were engineered, as previously described (Cova et al., 2015) and tested on FLCs from three SSc-ILD patients. We evaluated: apoptosis (Annexin V), proliferation (CFSE) and viability (MTT test). As control, functionalized GNPs without imatinib (GNP-HC) or imatinib alone (IM) were used. Only functionalized GNPs entered into FLCs (confocal microscopy). GNP-HCim inhibited cell proliferation at 48 and 72 h (p

Recent evidence links miRNA dysregulation with fibrogenic disorders, including idiopathic pulmonary fibrosis (IPF), in which miR21 has been found significantly upregulated in fibroblast foci as well as in patient serum(Liu et al., 2010). miR34a upregulation has also been found in macrophages in the bleomycin model(Honeymanet al., 2013). It is not clear if this phenomenon is specific to IPF or is more generally involved in lung fibrotic processes.In this work we aimed to compare the dysregulation of these 2 miRNAs in IPF with other fibrotic lung disorders such as cryptogenetic organizing pneumonia (COP) and bronchiolitis obliterans syndrome (BOS). We have evaluated miR21 and miR34a expression profiles by in-situ hybridization (ISH) ona set of formalin-fixed/paraffin-embedded lung samples obtained from explants or surgical biopsies in patients with IPF (n=10), COP (n=10) and BOS (n=6) and on bleomycin-treated rat lungs at 3, 7, 14 and 21 days after administration.Both miR-21 and miR-34 were expressed in fibroblasts and myofibroblasts of IPF fibroblast foci, of COP alveolar plugs and of bronchial obstructive lesions of BOS samples. There was no difference in staining distribution and intensity among samples within each diseases group. At semiquantitative evaluation, mir-21 was strongly expressed in fibroblasts and epithelial cells in remodeled areas in all 3 disease groups and in rat lungs, starting from the 7 th day; mir-34a expression was weaker in fibroblasts than in epithelial cells.Our results suggest that miR21 and miR34 overexpression are generally involved in fibrotic lung processes and are not specific for IPF, in accordance with the current view of IPF as the fibrotic outcome of injuries of different nature.

A functional B cell subset, CD19+CD24highCD38high, has been shown to contribute to the maintenance of the fine equilibrium required for tolerance, reducing the inflammatory responses during autoimmune diseases or unresolved infections. Pivotal to B-reg function is IL-10, which inhibits proinflammatory cytokines, supports T-reg cell differentiation and inhibits of Th17 and Th1 cell. The role of Breg cells in human lung transplantation is not yet elucidated, thus we aimed to analyze long-term kinetics of this subset in a cohort of lung recipients and test its association to several clinical and pharmacological variables (IS, azithromycin, extracorporeal photo-apheresis, infections, CLAD etc). From Jan 2009 to dec 2014, 117 lung transplant recipients were submitted to an immunological follow up (I-FU) (median:70 months):complete peripheral immuno-phenotype, inclusive of CD19+CD24highCD38high Breg cells (1102 determinations were analyzed).Linear OR regression models for repeated measures were used to test the association between B-reg and relevant variables, adjusting for time from Tx. Variables resulted significantly associated toperipheral B-reg cell counts in univariate analysis were: use of mycophenolate mofetil: neg association 0.001; while a pos association was present with Bacterial (0.022)and Fungal (0.042) infections.No association between B-reg cell counts and CLAD occurrence, the use of m-Tor inhibitors, azithromycin or other treatments such as ECP was detected. We suggest that regulatory B cells play a limited role in long-term lung transplant acceptance, being marginally influenced only by mycophenolate-mofetil and by respiratory and extrarespiratory infections.

The role of CD4+CD25highCD127- Treg cells in solid-organ transplant acceptance has been already studied, and while for kidney and liver an inverse correlation with long term graft survival was found; in lung transplant field, in particular with respect to association or prediction of CLAD, results are still controversial. Our study aims to analyze long term kinetic of CD4+CD25highCD127- Treg cells and test its association to several clinical and pharmacological variables(IS, azithromycin, extracorporeal photo-apheresis, infections, CLAD etc). From jan 2009 to dec 2014, 137 lung transplant recipients were submitted to an immunological follow up (I-FU)(105.9 months (6.7-310.5)). I-FU consisted of a complete peripheral immuno-phenotype inclusive of CD4+CD25highCD127- T cell counts and FOXP3 expression (1943 determinations analyzed). Linear OR regression models for repeated measures were used to test the association between Treg and relevant variables, adjusting for time from Tx. Ordered logistic models for panel data were used to test the association between CLAD onset/progression and Treg in the previous three months. Among the variables significantly associated at peripheral Treg cell counts at bivariate analysis: azathioprine therapy(p=0.03), ECP(p

The pathogenesis of Bronchiolitis Obliterans Syndrome (BOS), the main clinical phenotype of chronic lung allograft dysfunction, is poorly understood. Recent studies suggest that epigenetic regulation of microRNAs might play a role in its development. In this paper we present the application of a complex computational pipeline to perform enrichment analysis of miRNAs in pathways applied to the study of BOS. The analysis considered the full set of miRNAs annotated in miRBase (version 21), and applied a sequence of filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs according to their potential involvement in BOS development. Dysregulation of two of the selected candidate miRNAs–miR-34a and miR-21 –was clearly shown in in-situ hybridization (ISH) on five explanted human BOS lungs and on a rat model of acute and chronic lung rejection, thus definitely identifying miR-34a and miR-21 as pathogenic factors in BOS and confirming the effectiveness of the computational pipeline.

Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disorder characterized by a pattern of Usual Interstitial Pneumonia where the presence of fibroblastic foci is the hallmark of the disease.In the present study, we analyzed the migration inhibitory factor (MIF) expression in lung tissue of IPF patients compared with healthy controls and organizing pneumonia (OP) patients focusing into MIF potential role in fibroblastic foci development.The immunohistochemical analysis was performed in 10 IPF patients (7 male), 3 OP patients (2 male), and 3 healthy controls (all male) using the streptavidin-biotin method (Dako).In IPF samples, MIF resulted overexpressed in the areas of active fibrosis and, in particular, in the alveolar epithelium, bronchiolar epithelium, and in the peripheral zones of fibroblastic foci. Bronchiolar epithelium from organizing pneumonia patients resulted only weakly positive for MIF while no evidence of MIF expression was reported for alveolar epithelium. In the control subject group, MIF was unexpressed except for a weak presence in the bronchiolar epithelium.In conclusion, MIF is a pleiotropic cytokine involved in the pathogenesis of IPF being mainly expressed in the areas of remodeling and active fibrosis, in bronchiolar and alveolar epithelium, and in the peripheral zone of fibroblastic foci.

We discuss the case of synchronous bilateral lung cancers which feature the same histological phenotype and a different EGFR mutational profile. Both histological and molecular characterizations were performed on specimens derived thorough CT-guided fine needle aspiration. A first-line chemotherapy was unsuccessful. Subsequent objective response to the EGFR inhibitor Erlotinib was clearly coherent with the sequencing data and the mutated nodule was effectively reduced (> 50%) after therapy, while the lesion assessed as EGFR wild type featured a slight response. This report has two relevant implications. It points out that in case of multiple malignant lesions at time of diagnosis, molecular profiling should be as extensive as possible and it might contribute to clarify the association between the lesions found. Besides the molecular analysis on cytology specimens could identify an accurate and safe diagnostic approach for clinical use.

Involvement of CD44 in LungTx rejection has been suggested (Todd,2014). Among different CD44 isoforms, v6 seems associated with metastatic progression in cancer (Weidle, 2011). In a previous work (Cova, 2014), we demonstrated that CD44 was highly expressed by cells invading bronchiolar lumen in biopsies of BOS pts and by mesenchymal cells (MCs) from BAL of pts affected by BOS. In addition, CD44 expression was higher in MCs isolated from pts with more severe BOS. Aim of this work is to assay the expression and functional role of CD44v6 in MCs isolated from stable, BOS 0p, BOS1 LTx pts.CD44v6 mRNA expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR) and levels of v6 were normalized vs. relative amount expressed by MCs from a stable patient. A3D8 antibody (2 ml/ml) directed against standard CD44 (CD44s) was added to culture medium of MCs from a stable and a BOS1 patient and effect on cell proliferation was evaluated after 72 h by CFSE incorporation. Proliferation of MCs from a stable patient was significantly (p

The TK receptor MET orchestates 9invasive growth9, a genetic program driving the metastatic process. Deregulation of MET signaling pathway occurs via different mechanisms: protein overexpression, gene amplification, mutations or rearrangements. While the role of MET mutations in NSCLC is not yet fully understood, MET amplification drives cell survival and MET-amplified cells are sensitive to MET inhibition. This study aimed to evaluate the occurrence of genetic lesions affecting MET locus on 7q31.1 in primary e metastatic NSCLCs and to decipher a distribution pattern based on disease stage and outcome. A cohort of 69 cases of NSCLC and their matched brain metastases were firstly screened for the expression MET by IHC. Genomic tumor DNA was then sequenced to identify either MET somatic mutations, whereas gene copy number was carried on through FISH analysis on FFPE slides. The results found were then matched with corresponding clinical data. We demonstrate that MET locus was mainly overexpressed in NSCLC metastatic sites. MET somatic mutations were found in 5 of the 54 cases (9%) analysed : in two cases the same mutation was present in both primary and metastatic tissue, whereas in the remaining three mutation affected only brain metastasis. MET amplification was associated to a worst outcome and a poor response to chemotherapy. MET activation was related to tumor radioresistance. (p

Engineered gold nanoparticles targeted with a specific antibody (GNP-HCe) to primary lung mesenchymal cells (MC) isolated from BOS patients and loaded with everolimus were able to specifically inhibit MC proliferation and increase apoptosis without stimulating the inflammatory response (Cova, 2014). The aim of the present work was to prove that GNP-HCe can be safely administered to mice by inhalation. Nanoparticles (NPs) distribution and toxicity in vivo were assayed by aerosolization of 50 µg of labeled GNP-HCe or GNP-HC (gold nanoparticles with the antibody without everolimus) for 30 min a day for 2 weeks. Control mice were treated with saline solution. NPs localization was evaluated in lungs, kidney, spleen and liver by near-infrared (NIR) light imaging technology. Electron microscopy analysis (TEM) was performed to assess NP lung distribution. Toxic effects on tissue were evaluated by hystology. Inflammation burst in lung was tested by IL-8-homologue, CXCL1/KC, in BAL by ELISA NIR showed that fluorescence was significantly higher (p Our data showed that NPs were not taken by epithelial cells and were not toxic for other organs. Moreover, their presence did not stimulate the local inflammatory response. This work confirms the feasibility of GNP-HCe to be used as novel therapeutic approach for patients affected by CLAD.

Cancers of unknown primary origin (CUPs) encompass metastatic tumours in which first diagnosis is made in absence of a clinically detectable primary lesion. The biological features and the genomic makeup of such tumours remain largely unexplored. Because the hallmark of CUPs is their precocious neoplastic spread, it is likely that one or more molecular pathways involved in the metastatic process are hyperactive in these tumours. Among them the genetic program named 9invasive growth9 orchestrates different biological activities and is driven by the MET receptor. A preliminary screen revealed that mutations affecting MET coding sequence occur with remarkably high frequency in CUPs compared to unselected cancers (30% vs 3%). We now focus on pleural tumors of unknown primary (plCUP), a highly malignat disease for which no actionable target have been identified so far. We identified 4 patients diagnosed and followed in our Institution . We obtained cancer cells from thoracentesis and analysed the mutational profile, the activity and functional properties of pro-invasive signals, with an emphasis on TK receptors. In pl-CUP MET seems to be significantly activated due to gene amplification. One patient carried a mutation in the MET downstream effector, KRAS. These preliminary findings provide new, clinically pertinent data that could be fully exploited to develop anti MET strategies towards undifferentiated neoplastic pleural diseases.

BOS is characterized by fibrotic bronchiolar obliteration. MicroRNAs (miRs), are small, non-coding, single stranded RNAs playing a role in many biological processes including EMT. Recent studies demonstrated a role of miRs in fibrotic disorders but evidence on BOS are lacking. We applied an SB approach and computationally identified and scored a limited panel of miRs potentially relevant in BOS. All pathways involved in BOS were included, enhanced using the ReNEs software, then list of intronic miRs was extracted. The initial panel of 54 candidates was scored (Coverage and ValCov scores) considering miR activity on pathways and previous validation. To validate SB analysis we selected, among miR with ValCov score >0.4, miR21 (2° rank: 0.047) that was assessed by ISH on 3 samples: 3 mesenchimal cell lines (MC) derived from BAL of BOS pts, 2 lung BOS grafts of orthotopic rat lung Tx model (outbread CD SPF/VAF) and one explanted human BOS lung. MC lines expressed high level of miR21 with slight variation among lines. In addition, while native rat lung had no miR21 expression, a high degree of miR21 was documented in myofibroblasts of the OB lesion developed 15 days after Tx. Finally a very high degree of miR21 was documented in OB lesions of the BOS lung, while normal human lung had minimal miR21 positivity

Aims: Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). Materials & methods: Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. Results & discussion: Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. Conclusion: These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction. Original submitted 24 July 2013; Revised submitted 15 October 2013

Knowledge of tumor mutational status has become a priority for effective NSCLC-tailored treatment. NSCLC diagnosis is more often reached through biopsy; thus, there is a clear need to implement for routine tumor molecular profiling on small cytological samples. This work aims to screen and compare the EGFR and KRAS mutational prevalence in fresh tumor cells and in corresponding routinely processed samples derived from trans-thoracic fine-needle aspiration. The latter currently represents the most appropriate diagnostic procedure in case of peripheral lesions, such as adenocarcinomas, which account for almost 40% of all NSCLCs and for the highest EGFR mutational rates.Two hundred and forty-four patients carrying peripheral lung masses underwent CT-guided aspiration. The obtained material was split, and a part was addressed to conventional histopathological analysis while the remaining one was stored at -20 °C. In case of confirmation of adenocarcinoma, tumor genomic DNA was extracted from both fresh and fixed material, and EGFR and KRAS sequencing was performed.We identified 136 adenocarcinomas; from 134, we could recover enough material for the study. A full match was demonstrated between EGFR/KRAS mutational prevalences through the two approaches tested. We found EGFR mutations in 13 patients (9.7%); 7 were females and 11 never or former smokers. KRAS mutations occurred in 20 (14.9%) patients. EGFR and KRAS mutations were mutually exclusive.Mutational screening on fresh cancer cells is an achievable, safe and cost-effective procedure which might allow routinely tumor molecular profiling as powerful integration of conventional histopathological analysis.

Purpose The presence of mesenchymal cells (MC), the primary source of fibrotic cells, has been described in BAL fluid of LTR patients as predictive of BOS onset (Badri et al, 2011). CD44 cell surface glycoprotein has been associated with an invasive fibroblast phenotype. Aim of this work was to assay the presence of CD44 in MC isolated from BAL of LTR patients with BOS to set up biocompatible functionalized drug-loaded nanoparticles (NP) and to prove their effectiveness to specifically inhibit cell proliferation. The expression of active mTOR, the target of the selected drug, everolimus €, was also determined. Methods and Materials MC isolated from 5 BOS pts (grade 0, 1 and 2) were characterized for CD44, CD34, CD45, CD90 and CD105 surface marker expression by flow cytometry and used for the experiments. The presence of mTOR was assayed by immunocytochemistry. Fluorescent labeled NP functionalized with an anti-CD44 antibody and loaded with E (CD44NPE) have been used to assess cell uptake by confocal microscopy, cell apoptosis/death and proliferation by flow cytometry. Results CD44 was expressed by 85-98% of cultured cells while mTOR was present in all cells. Cell lines were also positive for CD90 and, in lower percentage, for CD105, and negative for CD34 and CD45. CD44 was not expressed by normal alveolar epithelia. CD44NPE were shown to adhere and enter into the cells after 45 min incubation. Drug free NP, used as negative control, were completely inert. CD44NPE treated cells showed a significantly higher mean rate of annexin V expression at 8 h (17,4 % vs. 4,5%) and 24 h (42,15% vs. 5,3%) respect to control cells while mean 7AAD expression at 24 h was 4,1 vs. 1.3%. Likewise, cell proliferation was significantly inhibited at 24, 72, 96 and 120 h. As a corollary, we also demonstrated lack of a pro-inflammatory activity of drug-loaded NP. Conclusions These results demonstrate the effectiveness of targeted drug-loaded NP to inhibit MC and are highly promising in the view of developing a less toxic, local treatment of BOS.

Background Systemic Sclerosis (SSc) is an autoimmune fibrotic disorder characterised by collagen and extracellular matrix deposition in the skin and internal organs, such as the lung. Interstitial lung involvement (ILD) is frequent in SSc but effective therapies are lacking and ILD represents one of the most frequent causes of death. Gold nanoparticles (GNPs) have a great potential in biomedical applications among which drug-delivery. Previously, we proved that GNPs containing everolimus and functionalized with anti-CD44 antibody targeted to mesenchymal cells from patients with chronic lung allograft rejection (CLAD) specifically inhibited these cells. We proved that also fibroblastoid-like cells (FLCs) isolated from bronchoalveolar lavage (BAL) of ILD-SSc patients express CD44. Objectives we loaded GNPs with the drug imatinib (GNP-HCim) with the aim to specifically inhibit FLCs from ILD-SSc patients. Methods GNPs with imatinib and exposing an anti-CD44 antibody were engineered, as previously described (1). GNP-HCim were incubated 2h with FLCs from three patients with ILD-SSc. Cell apoptosis (Annexin V) and proliferation (CFSE) were evaluated at different times by flow cytometry. Cell viability was assessed by MTT test. As control, functionalized GNPs without imatinib (GNP-HC) or imatinib alone (IM) were used. Fluorescent GNP-HC were generated to assess cell uptake by confocal microscopy and flow cytometry. Statistical differences were evaluated using ANOVA by Graph Prism 5.0 program. Results Fluoresce experiments showed that only HC-functionalized nanoparticles entered FLCs within 1 h. The results showed that GNP-HCim inhibited cell proliferation without significant differences between the three cell lines. In particular, the effect of GNP-HCim was significant at 48 (p Conclusions We proved that specifically engineered GNPs significantly inhibit proliferation and induce apoptosis of FLCs from ILD-SSc patients, as already described for CLAD patients. FLCs treatment with imatinib alone was not effective as GNP-HCim demonstrating the superiority of the nanoparticles. Our results also suggest an unspecific effect of nanoparticles themselves which has to be further clarified before the possibility to use engineered nanoparticles to develop new pharmacological treatment for patients with pulmonary fibroproliferative disorders. References Cova E, Colombo M, Inghilleri S, et al. Nanomedicine 2015. Disclosure of Interest None declared

Lung cancer is the leading cause of death for solid tumors worldwide with an annual mortality of over one million. Lung carcinoma includes a series of different diseases which are roughly divided into two groups based on clinical and histo-pathological features: non-small cell lung cancer (NSCLC), accounting for almost 80% of lung cancer diagnosis and small cell lung cancer (SCLC) responsible for the remaining 20%. The NSCLC molecular profile has been deeply investigated; alterations in several oncogenes, tumor suppressor genes and transcription factors have been detected, mainly in adenocarcinomas. Dissection of such a complex scenario represents a still open challenge for both researchers and clinicians. MET, the receptor for Hepatocyte Growth Factor (HGF), has been recently identified as a novel promising target in several human malignancies, including NSCLC. Deregulation of the HGF/MET signaling pathway can occur via different mechanisms, including HGF and/or MET overexpression, MET gene amplification, mutations or rearrangements. While the role of MET mutations in NSCLC is not yet fully understood, MET amplification emerged as a critical event in driving cell survival, with preclinical data suggesting that MET-amplified cell lines are exquisitely sensitive to MET inhibition. True MET amplification, which has been associated with poor prognosis in different retrospective series, is a relatively uncommon event in NSCLC, occurring in 1-7% of unselected cases. Nevertheless, in highly selected cohorts of patients, such as those harboring somatic mutations of EGFR with acquired resistance to EGFR tyrosine kinase inhibitors, MET amplification can be observed in up to 20% of cases. Preclinical data suggested that a treatment approach including a combination of EGFR and MET tyrosine kinases could be an effective strategy in this setting and led to the clinical investigation of multiple MET inhibitors in combination with anti-EGFR agents. Results from ongoing and future trials will clarify the role of anti-MET molecules for the treatment of NSCLC and will provide insights into the most appropriate timing for their use. The present review recapitulates the current knowledge on the role of MET signaling in NSCLC mainly focusing on its implications in molecular diagnostic approach and on the novel targeted inhibitors.

Background. Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology characterized by interstitial fibrosis determining irreversible distortion of pulmonary architecture. Reactive oxygen species (ROS) and markers of oxidative stress play a pivotal role in human IPF pathology, possibly through induction of epithelial-mesenchymal transition (EMT).Methods. We investigated by immunohistochemistry, in UIP and COP tissue samples, the expression of most relevant markers of the molecular interplay involving RAGE, oxidant/antioxidant balance regulation, tissue nitrosylation, and mediators of EMT.Results. In both UIP and COP, the degree of RAGE expression was similarly high, while SODs and i-NOS, diffusely present in COP endoalveolar plugs, were almost absent in UIP fibroblast foci. A lower degree of tissue nitrosilation was observed in UIP than in COP.Conclusions. Fibroblast lesions of UIP and of COP share a similar degree of activation of RAGE, while antioxidant enzyme expression markedly reduced in UIP.

One of the main differential diagnostic issues in pulmonary large-cell carcinomas is that involving neuroendocrine (NE) and basaloid histotypes. The differential diagnosis of basaloid versus large-cell NE carcinoma requires immunohistochemical determination of NE markers because of morphological overlap between the 2 entities. The authors report a unique case of lung carcinoma with basaloid architecture and NE immunohistochemical features observed in a 64-year-old male smoker who underwent upper left lobectomy for a neoplastic stenosis of the lobar bronchus. The patient died 14 months after surgery. Histological examination showed multiple peripheral nodules of moderately enlarged neoplastic cells with irregular nuclei, with granular chromatin and frequent nucleoli, and diffuse in situ neoplasia involving bronchi, peribronchial glands, and small airways. Immunohistochemistry documented diffuse expression of CD56 in neoplastic cells and isolated cell groups immunoreactive for basal cell markers. The reported case was considered an as-yet-undescribed tumor showing both basaloid and NE differentiation.

The hypothesis that epithelial-mesenchymal transition (EMT) contributes to the formation of fibroblast foci (FF), which are the histological hallmark and the site of active disease progression of Idiopathic Pulmonary Fibrosis (IPF), has not yet received a conclusive demonstration. Cells undergoing EMT lose epithelial features and acquire mesenchymal markers and morphology. Cadherin expression switch (from E to N) is one of the first events in EMT. We investigated the immunohistochemical expression of E- and N-cadherin, vimentin, fibronectin, laminin-5-γ2, α-smooth muscle actin, and fibroblast-specific protein-1 involved in EMT in 20 IPF lung biopsies, focusing on metaplastic squamous cells of bronchial basal origin, positive for laminin-5-γ2 and ΔNp63/p40, that cover FF. The results were compared with organizing pneumonia, reactive squamous cell metaplasia of bronchiolar epithelia, and squamous cell carcinoma. Bronchiolar basal metaplastic cells in IPF partially lost E-cadherin and expressed vimentin and fibronectin. Hyperplastic pneumocytes in IPF and controls coexpressed E-cadherin and N-cadherin, and were weakly positive for lam5-γ2. Reactive squamous cell metaplasia did not show any mesenchymal markers. Squamous cell carcinoma only expressed lam5-γ2. In IPF lungs, we observed two epithelial cell populations with a different expression profile of markers involved in EMT. Although neither hyperplastic pneumocytes nor bronchial basal cells showed evidence of complete EMT, only the latter seem to be specific for UIP and might have a role in its development.

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-beta1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.

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