Observational Prospective Analysis of Biological Characteristics of Mesothelioma Patients

Study Overview
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Brief Summary
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This is an observational prospective analysis of biological characteristics of malignant mesothelioma (MM) patients. Frozen and paraffin-embedded tumor specimens and pleural effusion of patients will be collected and than will be analyzed with the following analyses: 1Purification of Tumour Associated Macrophages (TAM) and tumour cells from pleural effusions. 2.Mass spectrometry analysis of TAMs and tumor cells 3.Co focal microscopy analysis of macrophages, tumour cells and specimens derived from patients.

Detailed Description
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This is an observational prospective analysis of biological characteristics of malignant pleural mesothelioma (MPM) patients. Frozen and paraffin-embedded tumor specimens and pleural effusion of patients will be collected and than will be analyzed with the following analyses: Purification of TAMs and tumour cells from pleural effusions. Pleural effusions will be subjected to centrifugation in order to separate cellular and non-cellular components. Cell-free supernatant will be collected and subjected to further analysis for the identification and characterization of secreted proteins (ELISA, Western Blot). Pleural effusion cell components will be characterized by cytofluorimetry in order to evaluate leukocytes composition (e.g. macrophages, neutrophils, lymphocytes). Next, to isolate TAMs from the cellular components, the investigators will adopt a standard protocol currently performed in the investigators group to purify TAMs from ascites derived from ovarian carcinoma patients . Briefly, TAMs will be separated by their selective capacity to adhere in culture plates in serum-free conditions. After 1 hour of incubation, non-adherent cells (mainly tumour cells and other inflammatory cells) will be thoroughly washed off with jets of medium. After adherence, cells were rested for 1 hour in standard culture conditions and subsequently lysed to extract proteins. To isolate tumour cells from leukocytes, a cell sorting-based approach will be used. Briefly, inflammatory cells will be stained with lymphocyte common antigen (CD45) antibody and negative cells (tumour cells) will be separated by cell sorting. Next tumour cells will be grown and amplified in standard culture conditions. Mass spectrometry analysis of TAMs and tumor cells Generation of proteome and the phospho-proteomic maps of TAMs and tumour cells will be accomplished by quantitation of both the entire proteome and the phospho-proteome. Results obtained with TAMs will be compared to untreated, M1 and M2 polarized Monocytes-derived macrophages (M-DM), while tumor cells purified from patients' pleural effusion will be analysed with respect to human mesothelial cells. Protein samples obtained from the purified TAMs and tumour cells, derived from the patient's pleural effusions, will be extracted using lysis/acetone precipitation. Cell extracts will be subsequently subjected to trypsin digestion and the resulting peptides will be labelled with the iTRAQ reagents. Labelled peptides will be separated with the first dimension of the separation being strong cation exchange (SCX) chromatography. Fractions will be collected and phospho-enriched, when required. The pooled mixture of peptides will be submitted to a second dimension chromatographic separation, organized as a two-step process: a desalting/concentrating pre-column, and an analytical column for the separation. System is a nano-flow configuration, with direct interfacing to a mass spectrometer. Tandem mass spectrometry will be performed on a hybrid ion-trap (IT) - Fourier transform ion-cyclotron-resonance mass spectrometer. Quantitative and identification data analysis will be carried out with Proteome Discoverer software (Thermo), using SEQUEST as the search engine. Statistical analysis and data classification will be carried out with aid of in-house written script for the freeware statistical analysis package. Co focal microscopy analysis of macrophages, tumour cells and specimens derived from patients. The analysis will be conducted on the cellular components derived from pleural effusion (as previously described) and from frozen and paraffin-embedded tissues. Immunofluorescence staining will be performed with markers of DNA damage and polarized inflammation. Furthermore, immunofluorescence analysis will be held to validate putative new MM molecular markers obtained from the mass spectrometry studies.

Official Title
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Diagnostic and Prognostic Role of New Inflammation Biomarkers in Patients With Malignant Pleural Mesothelioma

Conditions
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Malignant Mesothelioma

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Malignant pleural mesothelioma patients Availability of plural effusion and frozen and paraffin-embedded tumor specimens. Exclusion Criteria: Unavailability of plural effusion and frozen and paraffin-embedded tumor specimens.
Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| no treatment<br>no treatment | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| dissect the molecular mechanisms underlying the development of malignant mesothelioma | characterization of the microenvironment (expression of M1 and M2 markers of polarized inflammation - cytokines and chemokines inducers or inhibitors of the inflammatory response) and the interplay between tumour cells and TAMs (expression of DNA damage markers) in order to dissect the molecular mechanisms underlying the development of malignant mesothelioma | 24 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| study of the effect of TAMs | the study of the effect of TAMs on the induction of the DNA damage on (Human mast cell) HMC cells, and a possible synergic effect with asbestos fibers; | 24 months | 
| investigation of molecular mechanisms linking macrophages | the investigation of molecular mechanisms linking macrophages polarized activation with asbestos-induced mesothelioma | 24 months | 
| molecular characterization (proteome) of TAMs and tumor cells | the molecular characterization (proteome) of TAMs and tumor cells, isolated from pleural effusion derived from patients | 24 months | 
| compare in specimens from mesothelioma patients and the candidate molecular markers | to compare in specimens from mesothelioma patients and the candidate molecular markers, obtained expression in the above-mentioned tasks | 24 months | 
| Investigate the association of selected markers with Overall Survival | To Investigate the association of selected markers with Overall Survival (OS) | 24 months |