The combination of sorafenib and everolimus shows antitumor activity in preclinical models of malignant pleural mesothelioma.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify 'druggable' pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus. METHODS: We planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in 'hot spot' regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA). RESULTS: Phosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice. CONCLUSIONS: ERM and mTOR pathways are activated in MPM and 'druggable' by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.

Caveolin-1 as a promoter of tumour spreading: when, how, where and why.

Caveolae are non-clathrin invaginations of the plasma membrane in most cell types; they are involved in signalling functions and molecule trafficking, thus modulating several biological functions, including cell growth, apoptosis and angiogenesis. The major structural protein in caveolae is caveolin-1, which is known to act as a key regulator in cancer onset and progression through its role as a tumour suppressor. Caveolin-1 can also promote cell proliferation, survival and metastasis as well as chemo- and radioresistance. Here, we discuss recent findings and novel concepts that support a role for caveolin-1 in cancer development and its distant spreading. We also address the potential application of caveolin-1 in tumour therapy and diagnosis.

MET mutations in cancers of unknown primary origin (CUPs).

Cancer of unknown primary origin (CUP) defines metastatic disease of unknown origin, accounting for 3-5% of all cancers. Growing evidence demonstrates that inappropriate execution of a genetic program named "invasive growth," driven by the MET oncogene, is implicated in the metastatic process. MET activation in cancers is mainly consequent to overexpression, whereas mutations are rarely found. We reasoned that the occurrence of MET somatic mutations might sustain premature occult dissemination of cancer cells, such as that observed in CUPs. We sequenced MET in genomic DNA obtained from 47 early metastatic cancers. By extensive immunohistochemical analysis a primary site was afterward postulated in 24 patients, whereas 23 cases remained of unknown primary (CUPs). MET somatic mutations were found in seven cases, all belonging to the CUP cohort. Mutational incidence (30%) was thus significantly higher than the expected one (4%), in the absence of high mutational background. Several nucleotide changes were novel and clustered either in the kinase domain or in the extracellular semaphorin domain. Mutated receptors were functional and sustained the transformed phenotype, suggesting that MET activating mutations are genetic markers associated with the CUP syndrome.

Relationship between diffuse pulmonary fibrosis, alveolar proteinosis, and granulocyte-macrophage colony stimulating factor autoantibodies.

Extensive pulmonary fibrosis is a rare occurrence in pulmonary alveolar proteinosis. We report 2 cases that have interesting implications. A female patient was diagnosed with autoimmune pulmonary alveolar proteinosis that evolved over 7 years into diffuse fibrosis. In a male patient with diffuse fibrosis we incidentally detected electron microscopic features of alveolar surfactant accumulation and positive autoantibodies to granulocyte-macrophage colony stimulating factor. In the male patient we speculated that the pulmonary fibrosis might have been preceded by an asymptomatic phase of autoimmune pulmonary alveolar proteinosis, and that we should investigate the involvement of surfactant dysfunction in the pathogenesis of fibrotic lung disease.

Rescue use of beclomethasone and albuterol in a single inhaler for mild asthma.

BACKGROUND: Treatment guidelines recommend the regular use of inhaled corticosteroids for patients with mild persistent asthma. We investigated whether the symptom-driven use of a combination of beclomethasone dipropionate and albuterol (also known as salbutamol) in a single inhaler would be as effective as the regular use of inhaled beclomethasone and superior to the as-needed use of inhaled albuterol. METHODS: We conducted a 6-month, double-blind, double-dummy, randomized, parallel-group trial. After a 4-week run-in, patients with mild asthma were randomly assigned to receive one of four inhaled treatments: placebo twice daily plus 250 microg of beclomethasone and 100 microg of albuterol in a single inhaler as needed (as-needed combination therapy); placebo twice daily plus 100 microg of albuterol as needed (as-needed albuterol therapy); 250 microg of beclomethasone twice daily and 100 microg of albuterol as needed (regular beclomethasone therapy); or 250 microg of beclomethasone and 100 microg of albuterol in a single inhaler twice daily plus 100 microg of albuterol as needed (regular combination therapy). The primary outcome was the morning peak expiratory flow rate. RESULTS: In 455 patients with mild asthma who had a forced expiratory volume in 1 second of 2.96 liters (88.36% of the predicted value), the morning peak expiratory flow rate during the last 2 weeks of the 6-month treatment was higher (P=0.04) and the number of exacerbations during the 6-month treatment was lower (P=0.002) in the as-needed combination therapy group than in the as-needed albuterol therapy group, but the values in the as-needed combination therapy group were not significantly different from those in the groups receiving regular beclomethasone therapy or regular combination therapy. The cumulative dose of inhaled beclomethasone was lower in the as-needed combination therapy group than in the groups receiving regular beclomethasone therapy or regular combination therapy (P<0.001 for both comparisons). CONCLUSIONS: In patients with mild asthma, the symptom-driven use of inhaled beclomethasone (250 microg) and albuterol (100 microg) in a single inhaler is as effective as regular use of inhaled beclomethasone (250 microg twice daily) and is associated with a lower 6-month cumulative dose of the inhaled corticosteroid. (ClinicalTrials.gov number, NCT00382889 [ClinicalTrials.gov].).

Long-term outcome after pulmonary endarterectomy.

RATIONALE: There are few follow-up studies on long-term cardiopulmonary function after pulmonary endarterectomy (PEA), the operation of choice for chronic thromboembolic pulmonary hypertension (CTEPH). OBJECTIVES: To prospectively evaluate long-term outcome of patients with CTEPH treated with PEA. METHODS: Between 1994 and 2006, 157 patients (mean age 55 yr) were treated with PEA at Pavia University Hospital. The patients were evaluated before PEA and at 3 months (n = 132), 1 year (n = 110), 2 years (n = 86), 3 years (n = 69), and 4 years (n = 49) afterward by NYHA class, right heart hemodynamic, spirometry, carbon monoxide transfer factor (Tl(CO)), arterial blood gas, and treadmill incremental exercise test. MEASUREMENTS AND MAIN RESULTS: Cumulative survival was 84%. Within 3 months, 18 patients died in-hospital and 2 had lung transplantation; during long-term follow-up, 6 died, 1 had lung transplantation, and 3 had a second PEA (2.5 events per 100 person-years). NYHA class III-IV was the most important predictor of late death, lung transplant, or PEA redo (hazard ratio, 3.94). Extraordinary improvement in NYHA class, hemodynamic, and Pa(O(2)) were achieved in the first 3 months (P < 0.001) and persisted during follow-up; exercise tolerance progressively increased over time (P < 0.001). At 4 years, although 74% of the patients were in NYHA class I and none was in class IV, 24% had pulmonary vascular resistance greater than 500 dyne.s/cm(5) or Pa(O(2)) less than 60 mm Hg; they were significantly older and were more frequently in NYHA class III-IV 3 months after surgery than the others. CONCLUSIONS: After PEA, long-term survival and cardiopulmonary function recovery is excellent in most patients.

[AGE receptor: a novel pro-inflammatory pathway for chronic respiratory disorders?]. / RAGE: una nuova via pro-infiammatoria per le polmoniti interstiziali?

Interstitial pneumonites represent a heterogeneous group of respiratory disorders with known causes or idiopathic, in which alveolar injury is followed by defense and repair mechanisms either with pro- and anti-inflammatory properties. It is hypothetised that common factors would orchestrate such events, and RAGE is one of possible such candidates. Implications connected to RAGE pathway in varying interstitial pneumonites are discussed.

Foxp3 expressing CD4+ CD25+ and CD8+CD28- T regulatory cells in the peripheral blood of patients with lung cancer and pleural mesothelioma.

The role of T regulatory (Treg) cells in human cancer has not yet been clarified. We assessed the presence and function of CD4+ and CD8+ Treg cell subsets in the peripheral blood of patients with lung cancer (LC) and pleural mesothelioma (PM). We found a low but significant increase in the number of CD4+ T cells with phenotype and functional features of Treg cells in LC patients compared to normal healthy controls (NHC). Furthermore, total CD4+ T cells from LC patients proliferated less than cells from controls, suggesting that the increase in the CD4+ Treg cell pool has functional importance. LC patients also showed an expansion of the CD8+CD28- T cell subset and these cells expressed Foxp3 mRNA, as recently observed in alloantigen-specific CD8+CD28- T suppressor cells. No variation of peripheral Treg cell subsets was found in patients with PM, a disease with a predominantly localized nature. However, the lack of correlation between cancer stage and the number or the function of peripheral Treg cells in LC patients refuted the hypothesis that these cells are involved in tumor spreading. A possible involvement of the peripheral Treg cell pool in cancer development and/or in inducing systemic immunosuppression in LC patients can be hypothesized.

SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase.

BACKGROUND: Alpha1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind alpha1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction. METHODS AND RESULTS: At an alpha1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of alpha1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of alpha1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing alpha1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the alpha1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of alpha1-antitrypsin. CONCLUSION: We conclude that the binding of SP-A to alpha1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of alpha1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

NOD2/CARD15 gene polymorphisms in idiopathic pulmonary fibrosis.

BACKGROUND: Micro-organisms, behaving in a non-infectious fashion, may be among the exogenous factor(s) believed to trigger idiopathic pulmonary fibrosis (IPF). One possible strategy to identify an individual's susceptibility to such microbial triggers, which are likely to be ubiquitous, is to investigate the molecular processes involved in their recognition. NOD2/CARD15 is a specific pattern recognition receptor protein, whose genetic variants have been previously associated with susceptibility to Crohn's disease. AIM: The aim of this work was to determine the frequencies of the three major NOD2/CARD 15 gene mutations (R702W, G908R and 1007fsinC) in a series of 76 subjects affected by IPF, and to compare them with those found in three groups of controls: a group with sarcoidosis (a disorder in which an involvement of the NOD2/CARD15 gene has already been investigated and rejected in different ethnic groups; 67 subjects) and two groups of healthy subjects (218 and 208 subjects, respectively), matched for gender, age, and ethnicity. RESULTS: We found no differences in frequencies of NOD2/CARD15 gene polymorphisms among the four groups investigated. CONCLUSION: We conclude that the NOD2/CARD15 gene is not likely to be involved in susceptibility to IPF in Italians.

Exacerbations as a starting point of pro-active chronic obstructive pulmonary disease management.

Chronic obstructive pulmonary disease (COPD) exacerbations could represent an opportunity for pro-active COPD management rather than mere treatment if previously unknown disease is discovered; the extent of underdiagnosis and undertreatment of COPD in patients attending an emergency department (ED) with an exacerbation is not known. During 2002, we recalled 131 COPD patients in stable conditions, 4-8 weeks after they had attended the ED or been discharged from our University Hospital (North-West of Italy). Information on diagnosis and management prior to the ED attendance were collected; spirometry and arterial blood gas analyses were performed. One-third of patients had never been diagnosed and treated even though 83% of them had moderate-to-very-severe COPD and about 30% already had respiratory failure. Only 20% had received information on the nature of the disease and none had received a written action plan. Only 60% were receiving long-acting bronchodilators and 41% of patients with respiratory failure were receiving long-term oxygen. A substantial number of undiagnosed and untreated patients with moderate-to-very-severe COPD came to our attention through an exacerbation. This enforces the importance of exacerbations as the starting point of pro-active COPD management and of the ED as a valuable sentinel to identify this subset of patients.

Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients: correlation with transplant outcome.

BACKGROUND: The subset of CD4+CD25+ regulatory T cells, recently identified in humans, may play a central role in the regulation of immune tolerance to graft survival. METHODS: This study assesses the frequency and functional profile of CD4+CD25+CD69- cells in the peripheral blood of lung transplant recipients (>3 years from transplantation), 10 of whom were in a stable clinical condition and 11 of whom demonstrated chronic rejection (bronchiolitis obliterans syndrome). We also studied a group of seven healthy subjects. RESULTS: The frequency of CD4+ T cells expressing CD25 (CD4+CD25+) and the highest levels (CD25) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P < or = 0.01). Purified CD4+CD25+ cells exhibited a regulatory functional profile in vitro: they were hyporesponsive, suppressed the proliferation of CD4+CD25- cells, and produced interleukin-10. CONCLUSION: These results provide in vivo evidence that peripheral CD4+CD25+ T cells may represent an important regulatory subset in lung transplantation.

Assessment of emphysema in COPD: a functional and radiologic study.

OBJECTIVES: A combination of functional measurements reflecting a decrease in maximum flow, a degree of lung hyperinflation, the relationship between maximum inspiratory and expiratory flows, bronchodilator response, and diffusing capacity of the lung for carbon monoxide (DLCO) was used to quantify the extent of emphysema, as assessed by high-resolution CT (HRCT) scanning. DESIGN: Forced inspiratory and expiratory spirometry, lung volumes, reversibility test, and single-breath diffusing capacity were assessed before and after inhaling albuterol, 200 microg. Relationships between lung function variables and emphysema extent, as determined by HRCT scanning, were tested by univariate and multivariate analyses. SUBJECTS: Thirty-nine COPD outpatients with moderate-to-severe obstruction. MEASUREMENTS AND RESULTS: Emphysema extent, as assessed by HRCT scanning, ranged from 18 to 70%. All of the lung function parameters that were studied, except for the change in FEV1 percent predicted after salbutamol inhalation, correlated significantly with the extent of emphysema (r2 range, 0.19 to 0.44). Functional residual capacity, forced expiratory flow at 50% of FVC/forced inspiratory flow at 50% of FVC, DLCO/alveolar volume ratio, and bronchodilator-induced change in FEV1/FVC ratio were the only variables retained by stepwise multiple regression analysis. The multiple regression model explained 71% of the variability of emphysema extent measured by HRCT scanning. CONCLUSIONS: The combination of lung function measurements reflecting lung hyperinflation, bronchial collapsibility, lung diffusing capacity, and bronchodilator response provides a good estimate of the extent of emphysema, as evaluated by HRCT scanning. These data suggest that pulmonary function tests are useful in assessing and monitoring parenchymal damage in COPD patients.

BAL cytokine profile in different interstitial lung diseases: a focus on systemic sclerosis.

BACKGROUND AND AIM: Fibrosing alveolitis develops in up to 80% of systemic sclerosis patients (SSc) but progression to end stage fibrosis occurs in about 15% of cases. Mechanisms leading to the process remain mostly unknown. We compared cytokine profiles of broncho-alveolar lavage fluids (BAL-f) from patients with SSc associated interstitial lung disease (SSc-ILD) (n. 34), idiopathic pulmonary fibrosis (IPF) (n. 13), stage II sarcoidosis (n. 14) and 9 controls. METHODS: Interleukin (IL) 8, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon (IFN-gamma), IL12, IL18 and IL10 and transforming growth factor-beta (TGF-beta) were assessed by ELISA in concentrated BAL-f. RESULTS: Levels of IL8 and MCP-1 were significantly elevated in SSc-ILD and in IPF as compared with controls (Mann Whitney test p < 0.05), while MCP-1 values were significantly lower in SSc-ILD than in IPF. A significant correlation between neutrophils and IL8 levels (p = 0.047), as well as between eosinophils and MCP-1 levels (p = 0.004) was also observed. IFN-gamma levels were slightly higher than normal only in sarcoidosis (p = 0.06), whereas IL12 levels increased both in sarcoidosis and SSc-ILD (p < 0.05). No differences were found in IL18 and TGF-beta levels. Finally, IL10 levels were higher in SSc-ILD and sarcoidosis than in controls and IPF (p < 0.05). CONCLUSION: BAL-f cytokine profile differentiates ILD associated with SSc from IPF. The lower expression of MCP-1 and the higher expression of the anti-fibrotic IL12 and the anti-inflammatory IL10, observed both in sarcoidosis and in SSc-ILD, could account for the better prognosis of these ILDs. Further longitudinal studies are required to confirm whether a different cytokine phenotype may be considered predictive of clinical outcome in SSc-ILD.

Oncogenes in non-small-cell lung cancer: emerging connections and novel therapeutic dynamics.

Non-small-cell lung cancer is a heterogeneous disease that is difficult to treat. Through efforts to define the molecular mechanisms involved in lung oncogenesis, molecularly targeted approaches for patients with lung cancer have now reached the clinical arena. Despite elucidation of some molecular mechanisms of lung carcinogenesis, prognosis for patients remains poor. This Review aims to highlight the functional associations between key oncogenes that drive lung tumorigenesis and are distinct targetable molecules. Oncogenes are defined by acquisition of mutations, which results in a dominant gain-of-function of the targeted protein. In this situation, a single mutated allele is sufficient to induce malignant transformation. Importantly, tumours become addicted to particular genetic alterations that cause oncogene activation and the continued expression of the signalling. An increasing amount of evidence sustains the rationale for targeting of oncogenic pathways rather than a single oncogene. A clear priority for both researchers and clinicians is to better understand the complexity of biological networks underlying lung cancer pathogenesis. This paradigmatic shift in tailoring therapies should effectively improve outcomes for patients.

Non-neuronal cholinergic system in airways and lung cancer susceptibility.

In the airway tract acetylcholine (ACh) is known to be the mediator of the parasympathetic nervous system. However ACh is also synthesized by a large variety of non-neuronal cells. Strongest expression is documented in neuroendocrine and in epithelial cells (ciliated, basal and secretory elements). Growing evidence suggests that a cell-type specific Ach expression and release do exist and act with local autoparacrine loop in the non-neuronal airway compartment. Here we review the molecular mechanism by which Ach is involved in regulating various aspects of innate mucosal defense, including mucociliary clearance, regulation of macrophage activation as well as in promoting epithelial cells proliferation and conferring susceptibility to lung carcinoma onset. Importantly this non-neuronal cholinergic machinery is differently regulated than the neuronal one and could be specifically therapeutically targeted.

EGFR and KRAS mutational profiling in fresh non-small cell lung cancer (NSCLC) cells.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Targeting EGFR in non-small-cell lung cancer: lessons, experiences, strategies.

Cancer is a genetic disease and this concept is now widely exploited by both scientists and clinicians to design new targeted molecules. Indeed many data have already allowed us to ameliorate not only our knowledge about cancer onset, but also about patients treatment. Correlation between mutations in cancer alleles and drug response is a key point to identify drugs that match the genetic profile of each individual tumors. On the other hand, experience derived from inhibition of tyrosine kinase receptors has pointed out that targeted treatment is really successful only in a small subset of tumors. The latter are eventually addicted to those genetic alterations which are responsible for receptors activation and for the continued expression of their signalling. Overall these observations provide a strong rationale for a molecular-based diagnosis and patients selection for targeted therapies. This review analyses the current state of the art of molecularly-tailored pharmacological approach to lung cancer, one of the biggest killers among human solid tumors. Main relevance is addressed to genetic lesions activating the EGFR pathway transducers, focusing on their role as markers of targeted drug response.

Synchronous lung cancers: when same histological types feature different molecular profiles and response phenotypes.

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.

Factors influencing oxidative imbalance in pulmonary fibrosis: an immunohistochemical study.

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.