Observational Prospective Analysis of Biological Characteristics of Mesothelioma Patients

September 8, 2022 updated by: Armando Santoro, MD

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

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.

Study Overview

Status

Completed

Conditions

Detailed Description

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:

  1. 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.
  2. 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.
  3. 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.

Study Type

Observational

Enrollment (Actual)

100

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Italy
    • Milan
      • Rozzano, Milan, Italy, 20089
        • Istituto Clinico Humanitas
    • Milano
      • Rozzano, Milano, Italy, 20089
        • Istituto Clinico Humanitas

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Child
  • Adult
  • Older Adult

Accepts Healthy Volunteers

No

Genders Eligible for Study

All

Sampling Method

Probability Sample

Study Population

Malignant pleural mesothelioma patients

Description

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.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Observational Models: Case-Only
  • Time Perspectives: Prospective

Cohorts and Interventions

Group / Cohort
no treatment

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
dissect the molecular mechanisms underlying the development of malignant mesothelioma
Time Frame: 24 months
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
Time Frame: 24 months
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
Time Frame: 24 months
the investigation of molecular mechanisms linking macrophages polarized activation with asbestos-induced mesothelioma
24 months
molecular characterization (proteome) of TAMs and tumor cells
Time Frame: 24 months
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
Time Frame: 24 months
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
Time Frame: 24 months
To Investigate the association of selected markers with Overall Survival (OS)
24 months

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Armando Santoro, MD

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start

April 1, 2011

Primary Completion (Actual)

December 1, 2017

Study Completion (Actual)

January 1, 2018

Study Registration Dates

First Submitted

May 27, 2013

First Submitted That Met QC Criteria

November 24, 2014

First Posted (Estimate)

November 25, 2014

Study Record Updates

Last Update Posted (Actual)

September 9, 2022

Last Update Submitted That Met QC Criteria

September 8, 2022

Last Verified

September 1, 2022

More Information

Terms related to this study

Additional Relevant MeSH Terms

Other Study ID Numbers

  • ONC/OSS-02/2011

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

No

IPD Plan Description

not planned

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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