Ignite Creation Date:
2024-05-06 @ 10:21 AM
Last Modification Date:
2024-10-26 @ 12:28 PM
Study NCT ID:
NCT03233659
Status:
COMPLETED
Last Update Posted:
2023-02-01
First Post:
2017-07-26
Brief Title:
Immune Monitoring After Allogeneic Hematopoietic Stem Cell Transplantation
Sponsor:
IRCCS Azienda Ospedaliero-Universitaria di Bologna
Organization:
IRCCS Azienda Ospedaliero-Universitaria di Bologna
Study Overview
Official Title:
Study of the Function of Immune System in Patients Undergoing Allogeneic Stem Cell Transplantation
Status:
COMPLETED
Status Verified Date:
2023-01
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:
None
Brief Summary:
Graft versus Host disease GVHD is one of the major complications of Allogeneic Stem Cell Transplantation Acute GVHD develops early within 2to 3 months after transplantation and is the leading cause of death of transplanted patients The pathogenesis of Chronic GVHD is still little known Chronic GVHD is caused by donor T lymphocytes but we have no precise knowledge on the participation of specific subsets of immune system cells to chronic GVHD In general chronic GVHD is associated with an increase in the number of T effector lymphocytes both helper type 2 and cytotoxic
Recently also antigen presenting cells APCs have been implicated in pathogenesis of chronic GVHD in studies performed on animal models T lymphocyte responses that characterize chronic GVHD require that recipient antigens are submitted by APCs which originate from the donors HSC Hematopoietic Stem Cells APCs are heterogeneous population that includes dendritic cells DCs monocytes activated B lymphocytes and CD34 cell subpopulations These cells can be identified by cytometry
The data about APCs role in chronic GVHD are preliminary and often discordant Seemingly there isnt correlation between circulating APCs number and risk of cGVHD However recent data of our group show that patients with cGVHD could have higher number of monocytes in bone marrow than transplanted patients without cGVHD
The aim of study is to measure the number of circulating immune cells in the PB peripherical blood before and after Allogeneic Hematopoietic Stem Cell Transplantation by flow cytometry
Recently also antigen presenting cells APCs have been implicated in pathogenesis of chronic GVHD in studies performed on animal models T lymphocyte responses that characterize chronic GVHD require that recipient antigens are submitted by APCs which originate from the donors HSC Hematopoietic Stem Cells APCs are heterogeneous population that includes dendritic cells DCs monocytes activated B lymphocytes and CD34 cell subpopulations These cells can be identified by cytometry
The data about APCs role in chronic GVHD are preliminary and often discordant Seemingly there isnt correlation between circulating APCs number and risk of cGVHD However recent data of our group show that patients with cGVHD could have higher number of monocytes in bone marrow than transplanted patients without cGVHD
The aim of study is to measure the number of circulating immune cells in the PB peripherical blood before and after Allogeneic Hematopoietic Stem Cell Transplantation by flow cytometry
Detailed Description:
Study Endpoints
The primary endpoint of study is detection of circulating cells by flow cytometry before and after Allogeneic Stem Cell Transplantation We analyze the following cells Myeloid Dendritic Cells Plasmacytoid Dendritic cells CD16 Dendritic cells CD14 monocytes CD14CD16 monocytes total T lymphocytes total T helper lymphocytes central memory naive effector memory and effector T helper lymphocytes total cytotoxic lymphocytes central memory naive effector memory and effector T cytotoxic lymphocytes CD16CD56 NK cells B lymphocytes T regulatory lymphocytes
The secondary endpoints are
Determination of the serum concentration of Cytokines TNFα IFNγ IL-4 and Chemokines MIP and MCP group
Determination of autoantibodies in the serum In the first phase screening analysis is performed by searching anti nuclear antibodies ANA and antibodies anti-ENA Extractable Nuclear Antigens Positive results are subjected to second level analysis Next we are proceed to the analysis of different Organ-Specific Autoantibodies previously associated with chronic GVHD like liver antibodies SMA AMA SLA anti-cardiolipin and beta2 microglobulin antibodies Antibodies against thyroid and gastric parietal cells
Study of CD86 CXCR4 CCR2 CCR5 CD11a and CD49d molecules expression in Monocytes and Dendritic Cells
Study of TNF and IL-12 production by purified monocytes
Study of Allostimulatory activity of purified monocytes against Allogeneic CD4 T cells
Study of antigen capture and processing by phagocytosis macropinocytosis and endocytosis mediated by purified monocytes receptors
Study of CD134 0X40L CD154 CD40L molecules expression study of differentiation molecules type1 and type 2 T helper cells and T regulatory Cells associated study of molecules Cellular Death associated
Study of Cytokines proliferation and production including IL-2 IL-5 IFNγ and IL-10 and release of perforin and granzyme after stimulation with mitogens
Study of antigen-specific T lymphocytes response by pentamers for detection of HY antigen by ELISPOT assay Recipients cells are collected and stored before the transplantation
PCR study of micro mi RNA in patients serum and cells
PCR and Microarray study of gene expression profile of transplanted monocytes
Study design
Observational single center prospective study employing human tissues for in vitro study after Allogeneic Stem Cell Transplantation The study includes a retrospective phase The study requires that patients Peripheral blood samples are harvested
on entering the department
after 1 month of transplantation
after 3 6 912 months of transplantation
Study Population
All patients undergoing Allogeneic Transplantation in our Institute are included
Collection and processing of blood samples
Each peripheral blood sample must be collected in three heparin tubes up to 18 ml of SP and a non-anticoagulant tube 6 ml for a total of at least 3 ml of serum
Each sample is processed as follows
Blood sample taken without anticoagulant will be centrifuged to obtain serum the aliquots of serum are frozen and stored at -20C The immunophenotypic analysis for number and function of the mentioned cells are performed on fresh PB samples within 72 hours of collection
Mononuclear cells and monocytes are purified by standard procedures MNCs by density gradient centrifugation monocytes by immunomagnetic selection and then stored in liquid nitrogen until they are used for expected functional assays The negative CD14 fraction are frozen and used as the source of T lymphocytes
Flow cytometry analysis
The number and phenotype of the immune system circulating cells are determinated by flow cytometry using the specific monoclonal antibodiesWe are considered the following immune cells
CD4 and CD8 T lymphocytes including T naïve lymphocytes CD45RA CCR7 effector memory lymphocytes CD45RA-CCR7- central memory lymphocytes CD45RA-CCR7 and effector lymphocytes CD45RA-CCR7
T regulatory lymphocytes CD4 CD25 CD127-FoxP3
B Lymphocytes CD19
NK cells CD56 CD16- and cytotoxic CD56 CD16 cells
Myeloid Dendritic Cells CD11c lineage- DR CD33 Plasmacytoid Dendritic Cells lineage- DR CD33- and CD123 and Monocytoid CD33 CD14- CD16 DCs
Inflammatory CD14 CD16 and Constituents CD14 CD16- Monocytes
Functional studies on purified monocytes
TNF alpha and IL12 production are measured in flow cytometry
The allostimulatory function is determined in mixed leukocyte cultures using HLA different CD4 T lymphocytes as responders
The ability to capture antigens is measured in flow cytometry on individual cells by fluorescence antigens such as FITC conjugated albumin or fluorescent beads
The gene expression profile is performed using a MICROARRAY study
Functional studies on purified T lymphocytes
The expression of activation differentiation and apoptosis molecules is determinated by cytometry
Cytokine production is determined in single-color flow cytometry after stimulation with anti-CD3 and anti-CD28
In positive male HLA-A2 patients receiving female donor transplantation the frequency of T anti-HY lymphocytes is measured by fluorochrome-conjugated pentamers
In all other patients T-cell donor response to receptor antigens is measured in ELISPOT
Serum cytokines Study
The concentration of serum cytokines is determined in cytometry using CBA Cytokine Bead Assay CBA Becton Dickinson Mountian View CA USA We analyze the following cytokines
T-helper 1 cytokines TNF-alpha IFN-gamma and IL-12
T-helper 2 and regulators cytokines IL-10 IL-5 and TGF-beta
Chemokines MCP1 MCP2 RANTES MIP1-alpha and MIP1-beta IL-8 IP-10
Serum autoantibodies Study
The concentration of the antinuclear antibodies is measured by indirect immunofluorescence on HEp2 cells a cell line of hepatocarcinoma The antibodies against extractable nuclear antigens ENA are measured by ELISA ENA SCREENING In case of positivity the presence of antibodies against specific antigens is determined by DOT BLOT
micromi RNA PCR study in patients serum and cells
The total RNA is extracted from human serum samples and stored at -80 C A part of RNA 3µl is processed for reverse transcription and pre-amplification with a pool of primers according to the suppliers description 664 human miRNA 6 small human RNA and 1 control miRNA are processed in parallel
Gene expression profile Study of transplanted patients monocytes by PCR and MICROARRAY
The monocyte RNA messenger is extracted from the cells converted into cDNA by reverse transcriptase and at the same time labeled with a fluorescent probe The hybridization between the nucleic acids probes present on the matrix and the cDNA target will remain hybridized and can then be identified by detecting the location where it is bound At the end of study all samples will be destroyed
Concomitant treatments
Patients will receive or have received chemotherapy for transplant preparation according to current clinical practice After transplantation patients will receive medication for prophylaxis of GVHD and infections according to clinical practice Concomitant therapies possibly administered according to clinical practice are documented in CRF
Schedule of visits and evaluations
Patient visit is expected and CRF is completed at the enrollment There are 6 blood samples per patient Any blood sample after the enrollment can be delayed or anticipated by 14 days
Laboratory Tests
Tests are performed under the normal care pathway and are not study-specific Blood exam informations are reported on CRFs
Statistical analysis
The primary endpoint of the study is the measure of circulating immune cell number in PB before and after Allogeneic Hematopoietic Stem Cell Transplantation Statistical analysis consists of comparison analysis between averages using GRAPH PAD PRISM version 402 program The level of significance is defined as 05 Analysis of secondary endpoints is performed using the same methods
Sample Size
The study is proposed to all patients undergoing allogeneic transplantation The number of transplants performed at the Seràgnoli Institute is on average between 50 and 60 per year It is therefore estimated that 300 patients will be enrolled in the study
Administrative procedures
Any modification to the protocol will be made in the form of the amendment No other types of modification to the protocol are allowed during the study period Any unexpected changes in the study will be recorded in the Clinical Study Report
Management of informed consent
All patients date and write the Informed Consent within one of the visits provided by the normal care path For dead patients it is considered that the data processing is authorized with the approval of the study by the Ethics Committee according to general authorization Official Journal 72 of 26032012
Documentation archive
The principal Investigator is responsible for storing of the essential documents of the study before during and after the completion or termination of the study in accordance with the time required by the applicable laws and GCPs
The data collected in the CRF are strictly anonymous and the subject is only identified with a number and initials
The Investigator will have to keep the patients original data and informed written consent
Inspections Audits
If a Regulatory Authority requires an inspection the Investigator must immediately inform the Ethics Committee
Publication of results
The results of the experiment will be published within twelve months of the conclusion
The primary endpoint of study is detection of circulating cells by flow cytometry before and after Allogeneic Stem Cell Transplantation We analyze the following cells Myeloid Dendritic Cells Plasmacytoid Dendritic cells CD16 Dendritic cells CD14 monocytes CD14CD16 monocytes total T lymphocytes total T helper lymphocytes central memory naive effector memory and effector T helper lymphocytes total cytotoxic lymphocytes central memory naive effector memory and effector T cytotoxic lymphocytes CD16CD56 NK cells B lymphocytes T regulatory lymphocytes
The secondary endpoints are
Determination of the serum concentration of Cytokines TNFα IFNγ IL-4 and Chemokines MIP and MCP group
Determination of autoantibodies in the serum In the first phase screening analysis is performed by searching anti nuclear antibodies ANA and antibodies anti-ENA Extractable Nuclear Antigens Positive results are subjected to second level analysis Next we are proceed to the analysis of different Organ-Specific Autoantibodies previously associated with chronic GVHD like liver antibodies SMA AMA SLA anti-cardiolipin and beta2 microglobulin antibodies Antibodies against thyroid and gastric parietal cells
Study of CD86 CXCR4 CCR2 CCR5 CD11a and CD49d molecules expression in Monocytes and Dendritic Cells
Study of TNF and IL-12 production by purified monocytes
Study of Allostimulatory activity of purified monocytes against Allogeneic CD4 T cells
Study of antigen capture and processing by phagocytosis macropinocytosis and endocytosis mediated by purified monocytes receptors
Study of CD134 0X40L CD154 CD40L molecules expression study of differentiation molecules type1 and type 2 T helper cells and T regulatory Cells associated study of molecules Cellular Death associated
Study of Cytokines proliferation and production including IL-2 IL-5 IFNγ and IL-10 and release of perforin and granzyme after stimulation with mitogens
Study of antigen-specific T lymphocytes response by pentamers for detection of HY antigen by ELISPOT assay Recipients cells are collected and stored before the transplantation
PCR study of micro mi RNA in patients serum and cells
PCR and Microarray study of gene expression profile of transplanted monocytes
Study design
Observational single center prospective study employing human tissues for in vitro study after Allogeneic Stem Cell Transplantation The study includes a retrospective phase The study requires that patients Peripheral blood samples are harvested
on entering the department
after 1 month of transplantation
after 3 6 912 months of transplantation
Study Population
All patients undergoing Allogeneic Transplantation in our Institute are included
Collection and processing of blood samples
Each peripheral blood sample must be collected in three heparin tubes up to 18 ml of SP and a non-anticoagulant tube 6 ml for a total of at least 3 ml of serum
Each sample is processed as follows
Blood sample taken without anticoagulant will be centrifuged to obtain serum the aliquots of serum are frozen and stored at -20C The immunophenotypic analysis for number and function of the mentioned cells are performed on fresh PB samples within 72 hours of collection
Mononuclear cells and monocytes are purified by standard procedures MNCs by density gradient centrifugation monocytes by immunomagnetic selection and then stored in liquid nitrogen until they are used for expected functional assays The negative CD14 fraction are frozen and used as the source of T lymphocytes
Flow cytometry analysis
The number and phenotype of the immune system circulating cells are determinated by flow cytometry using the specific monoclonal antibodiesWe are considered the following immune cells
CD4 and CD8 T lymphocytes including T naïve lymphocytes CD45RA CCR7 effector memory lymphocytes CD45RA-CCR7- central memory lymphocytes CD45RA-CCR7 and effector lymphocytes CD45RA-CCR7
T regulatory lymphocytes CD4 CD25 CD127-FoxP3
B Lymphocytes CD19
NK cells CD56 CD16- and cytotoxic CD56 CD16 cells
Myeloid Dendritic Cells CD11c lineage- DR CD33 Plasmacytoid Dendritic Cells lineage- DR CD33- and CD123 and Monocytoid CD33 CD14- CD16 DCs
Inflammatory CD14 CD16 and Constituents CD14 CD16- Monocytes
Functional studies on purified monocytes
TNF alpha and IL12 production are measured in flow cytometry
The allostimulatory function is determined in mixed leukocyte cultures using HLA different CD4 T lymphocytes as responders
The ability to capture antigens is measured in flow cytometry on individual cells by fluorescence antigens such as FITC conjugated albumin or fluorescent beads
The gene expression profile is performed using a MICROARRAY study
Functional studies on purified T lymphocytes
The expression of activation differentiation and apoptosis molecules is determinated by cytometry
Cytokine production is determined in single-color flow cytometry after stimulation with anti-CD3 and anti-CD28
In positive male HLA-A2 patients receiving female donor transplantation the frequency of T anti-HY lymphocytes is measured by fluorochrome-conjugated pentamers
In all other patients T-cell donor response to receptor antigens is measured in ELISPOT
Serum cytokines Study
The concentration of serum cytokines is determined in cytometry using CBA Cytokine Bead Assay CBA Becton Dickinson Mountian View CA USA We analyze the following cytokines
T-helper 1 cytokines TNF-alpha IFN-gamma and IL-12
T-helper 2 and regulators cytokines IL-10 IL-5 and TGF-beta
Chemokines MCP1 MCP2 RANTES MIP1-alpha and MIP1-beta IL-8 IP-10
Serum autoantibodies Study
The concentration of the antinuclear antibodies is measured by indirect immunofluorescence on HEp2 cells a cell line of hepatocarcinoma The antibodies against extractable nuclear antigens ENA are measured by ELISA ENA SCREENING In case of positivity the presence of antibodies against specific antigens is determined by DOT BLOT
micromi RNA PCR study in patients serum and cells
The total RNA is extracted from human serum samples and stored at -80 C A part of RNA 3µl is processed for reverse transcription and pre-amplification with a pool of primers according to the suppliers description 664 human miRNA 6 small human RNA and 1 control miRNA are processed in parallel
Gene expression profile Study of transplanted patients monocytes by PCR and MICROARRAY
The monocyte RNA messenger is extracted from the cells converted into cDNA by reverse transcriptase and at the same time labeled with a fluorescent probe The hybridization between the nucleic acids probes present on the matrix and the cDNA target will remain hybridized and can then be identified by detecting the location where it is bound At the end of study all samples will be destroyed
Concomitant treatments
Patients will receive or have received chemotherapy for transplant preparation according to current clinical practice After transplantation patients will receive medication for prophylaxis of GVHD and infections according to clinical practice Concomitant therapies possibly administered according to clinical practice are documented in CRF
Schedule of visits and evaluations
Patient visit is expected and CRF is completed at the enrollment There are 6 blood samples per patient Any blood sample after the enrollment can be delayed or anticipated by 14 days
Laboratory Tests
Tests are performed under the normal care pathway and are not study-specific Blood exam informations are reported on CRFs
Statistical analysis
The primary endpoint of the study is the measure of circulating immune cell number in PB before and after Allogeneic Hematopoietic Stem Cell Transplantation Statistical analysis consists of comparison analysis between averages using GRAPH PAD PRISM version 402 program The level of significance is defined as 05 Analysis of secondary endpoints is performed using the same methods
Sample Size
The study is proposed to all patients undergoing allogeneic transplantation The number of transplants performed at the Seràgnoli Institute is on average between 50 and 60 per year It is therefore estimated that 300 patients will be enrolled in the study
Administrative procedures
Any modification to the protocol will be made in the form of the amendment No other types of modification to the protocol are allowed during the study period Any unexpected changes in the study will be recorded in the Clinical Study Report
Management of informed consent
All patients date and write the Informed Consent within one of the visits provided by the normal care path For dead patients it is considered that the data processing is authorized with the approval of the study by the Ethics Committee according to general authorization Official Journal 72 of 26032012
Documentation archive
The principal Investigator is responsible for storing of the essential documents of the study before during and after the completion or termination of the study in accordance with the time required by the applicable laws and GCPs
The data collected in the CRF are strictly anonymous and the subject is only identified with a number and initials
The Investigator will have to keep the patients original data and informed written consent
Inspections Audits
If a Regulatory Authority requires an inspection the Investigator must immediately inform the Ethics Committee
Publication of results
The results of the experiment will be published within twelve months of the conclusion
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