Ignite Creation Date:
2024-05-06 @ 7:05 PM
Last Modification Date:
2024-10-26 @ 3:00 PM
Study NCT ID:
NCT05889481
Status:
RECRUITING
Last Update Posted:
2023-06-18
First Post:
2023-05-26
Brief Title:
Morphological Genetic and Tumour Microenvironment Characterisation in Uveal Melanoma
Sponsor:
Fondazione Policlinico Universitario Agostino Gemelli IRCCS
Organization:
Fondazione Policlinico Universitario Agostino Gemelli IRCCS
Study Overview
Official Title:
Morphological Genetic and Tumour Microenvironment Characterisation in Uveal Melanoma and Impact on the Development of Metastasis and Mortality
Status:
RECRUITING
Status Verified Date:
2023-06
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
MicroGenUM
Brief Summary:
The objectives of the study are the morpho-phenotypic evaluation of uveal melanoma and to identify molecular prognostic factors that may be correlated with disease severity tumour progression and response to treatment
These objectives will be achieved through immunohistochemical and genetic analyses
These objectives will be achieved through immunohistochemical and genetic analyses
Detailed Description:
Tissues from surgical specimens regardless of the stage of the disease fixed in formalin and included in paraffin from samples already collected for clinical practice and kept in the archives of the UOC of Pathological Anatomy of the Fondazione Policlinico Universitario A Gemelli IRCCS will be used for the study subject to the patients informed consent to the use of these samples for research purposes The UOC Anatomia Patologica will take care of the recovery of archive material slides
The pseudoanonymised slides which will be prepared by clinical practice will be sent to the Institute of Pathological Histology and Molecular Diagnostics of the Azienda Ospedaliero- Universitaria Careggi Florence for immunohistochemical analysis which will be carried out as reported below Once the following analysis has been performed the slides will be destroyed
Sections of 3 μm thickness derived from FFPE samples will be prepared for immunohistochemical analysis Sample processing will be performed using an automated immunostainer Ventana Discovery ULTRA Ventana Medical Systems Tucson AZ Sections will be deparaffinised in EZ prep 950-102 Ventana Medical Systems Tucson AZ followed by treatment with Cell Conditioning 1 CC1 buffer 950-124 Ventana Medical Systems Tucson AZ in order to promote antigenic recovery Finally primary antibodies will be dispensed according to the desired staining and the signal will be developed with Ultramap anti-mouse or anti-rabbit detection kits For TME characterisation samples will be stained with the following primary antibodies CD3 CD4 CD8 CD68 CD163 All sections processed with IHC will be digitally scanned in Whole Slide Images WSI using the Aperio AT2 platform Leica Biosystems Wetzlar DEDigital Pathology which will be performed at the University of Florence is an emerging discipline that allows the quantitative analysis of digital images using highly standardised approaches It will allow quantitative measurements of the interactions between immune cells and characterise their relative spatial distribution In addition it will be able to generate further information that will allow the TME of the MU to be studied in depth
The HALO image analysis platform present at the University of Florence will allow i cell quantification evaluation of the intensity of the marking recognition of cell compartments nucleus cytoplasm membrane ii spatial analysis through the identification of the relative distributions of cells in the intra- and peri-tumour portion iii sharing of the database generated together with the digital images
Samples labelled with single or multiple IHC will be evaluated using this automated digital quantification system in order to characterise the various cell subsets that make up the TME By combining multiple immunohistochemistry with digital analysis the data will be accurately analysed thus producing a new analysis work-flow for finding new signatures All data derived from the digital analysis will be correlated with the progression of the pathology with the aim of identifying new prognostic factors in the pathology of MU
The analysis for the identification of cytogenetic alterations whose data are already available to us since they are regularly performed in clinical practice was conducted on the entire cohort of patients enrolled for the study at the Genetics Institute of the Fondazione Policlinico Gemelli on fresh tissue samples taken in the operating theatre as per clinical practice Array-CGH methods on oligonucleotide platforms from Agilent Technologies were used Tumour DNA from MU samples was extracted using the Gentra Pure Gene commercial kit QIAGEN as per clinical practice once extracted the quantity and purity of the DNA obtained was evaluated All samples deemed suitable for quality were processed following the protocol indicated by Agilent Technologies The data obtained were analysed using Cytogenomics software These analyses will allow a genotypic characterisation of the tumour to be obtained
The pseudoanonymised slides which will be prepared by clinical practice will be sent to the Institute of Pathological Histology and Molecular Diagnostics of the Azienda Ospedaliero- Universitaria Careggi Florence for immunohistochemical analysis which will be carried out as reported below Once the following analysis has been performed the slides will be destroyed
Sections of 3 μm thickness derived from FFPE samples will be prepared for immunohistochemical analysis Sample processing will be performed using an automated immunostainer Ventana Discovery ULTRA Ventana Medical Systems Tucson AZ Sections will be deparaffinised in EZ prep 950-102 Ventana Medical Systems Tucson AZ followed by treatment with Cell Conditioning 1 CC1 buffer 950-124 Ventana Medical Systems Tucson AZ in order to promote antigenic recovery Finally primary antibodies will be dispensed according to the desired staining and the signal will be developed with Ultramap anti-mouse or anti-rabbit detection kits For TME characterisation samples will be stained with the following primary antibodies CD3 CD4 CD8 CD68 CD163 All sections processed with IHC will be digitally scanned in Whole Slide Images WSI using the Aperio AT2 platform Leica Biosystems Wetzlar DEDigital Pathology which will be performed at the University of Florence is an emerging discipline that allows the quantitative analysis of digital images using highly standardised approaches It will allow quantitative measurements of the interactions between immune cells and characterise their relative spatial distribution In addition it will be able to generate further information that will allow the TME of the MU to be studied in depth
The HALO image analysis platform present at the University of Florence will allow i cell quantification evaluation of the intensity of the marking recognition of cell compartments nucleus cytoplasm membrane ii spatial analysis through the identification of the relative distributions of cells in the intra- and peri-tumour portion iii sharing of the database generated together with the digital images
Samples labelled with single or multiple IHC will be evaluated using this automated digital quantification system in order to characterise the various cell subsets that make up the TME By combining multiple immunohistochemistry with digital analysis the data will be accurately analysed thus producing a new analysis work-flow for finding new signatures All data derived from the digital analysis will be correlated with the progression of the pathology with the aim of identifying new prognostic factors in the pathology of MU
The analysis for the identification of cytogenetic alterations whose data are already available to us since they are regularly performed in clinical practice was conducted on the entire cohort of patients enrolled for the study at the Genetics Institute of the Fondazione Policlinico Gemelli on fresh tissue samples taken in the operating theatre as per clinical practice Array-CGH methods on oligonucleotide platforms from Agilent Technologies were used Tumour DNA from MU samples was extracted using the Gentra Pure Gene commercial kit QIAGEN as per clinical practice once extracted the quantity and purity of the DNA obtained was evaluated All samples deemed suitable for quality were processed following the protocol indicated by Agilent Technologies The data obtained were analysed using Cytogenomics software These analyses will allow a genotypic characterisation of the tumour to be obtained
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None