Brief Title
Observational Prospective Analysis of Biological Characteristics of Mesothelioma Patients
Official Title
Diagnostic and Prognostic Role of New Inflammation Biomarkers in Patients With Malignant Pleural Mesothelioma
Brief Summary
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
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
Primary Outcome
dissect the molecular mechanisms underlying the development of malignant mesothelioma
Secondary Outcome
study of the effect of TAMs
Condition
Malignant Mesothelioma
Study Arms / Comparison Groups
no treatment
Description: no treatment
Publications
* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.
Recruitment Information
Estimated Enrollment
100
Start Date
April 2011
Completion Date
January 2018
Primary Completion Date
December 2017
Eligibility Criteria
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.
Gender
All
Ages
N/A - N/A
Accepts Healthy Volunteers
No
Contacts
armando Santoro, MD, ,
Location Countries
Italy
Location Countries
Italy
Administrative Informations
NCT ID
NCT02300883
Organization ID
ONC/OSS-02/2011
Responsible Party
Sponsor-Investigator
Study Sponsor
Armando Santoro, MD
Study Sponsor
armando Santoro, MD, Principal Investigator, Istituto Clinico Humanitas
Verification Date
October 2017