Ignite Creation Date:
2024-05-06 @ 11:39 AM
Last Modification Date:
2024-10-26 @ 12:47 PM
Study NCT ID:
NCT03561402
Status:
COMPLETED
Last Update Posted:
2021-03-24
First Post:
2018-06-07
Brief Title:
Biomarkers and Disease Activity in Patients Treated With Teriflunomide Aubagio
Sponsor:
McGill University
Organization:
McGill University
Study Overview
Official Title:
Association of Possible Biomarkers With Disease Activity in Patients Treated With Teriflunomide Aubagio
Status:
COMPLETED
Status Verified Date:
2021-03
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:
The study is a two-year prospective observational study of patients treated with teriflunomide The investigators will recruit up to 75 patients at baseline based on the estimate that approximately 20 of these patients 15 patients will have evidence of disease activity at the end of the first year of treatment with teriflunomide as determined by clinical evaluation relapses and MRI activity new T2 hyperintense lesions The investigators will assess the expression of a putative biomarker signature consisting of toll like receptor 2TLR2 TLR4 and chemokine receptor 1 CCR1 on CD4 T-subsets at baseline and at intervals on treatment with teriflunomide to determine whether expression of this biomarker signature on one or more CD4 T-subsets correlates with disease activity
Detailed Description:
TITLE Association of possible biomarkers with disease activity in patients treated with teriflunomide Aubagio
A BACKGROUND Teriflunomide Aubagio is a once-daily oral immunomodulatory DMT for patients with relapsing-remitting MS RRMS 1 The objective of the present study is to determine whether a putative biomarker signature predicts disease activity in patients treated with teriflunomide The investigators previously identified a 130-gene signature associated with immune activation that identified patients with MS that had rapid transition to secondary progressive MS SPMS From this signature the investigators identified three genes TLR2 TLR4 and CCR1 which had increased protein expression on naïve CD4 T-cells in these patients The investigators also showed that mRNA for an anti-proliferation factor termed TOB1 was downregulated in these T-cells in patients with rapid MS progression The findings suggest therefore that naïve CD4 T-cell activation identifies patients with MS having a short RRMS duration 2
For this proposal the principal investigator suggests that various molecules involved in T-cell activation may also serve as useful biomarkers to predict treatment responses to teriflunomide
B STUDY OBJECTIVES
B1 Study objective and specific aims The objective is to determine whether the T-cell activation markers that were previously identified will predict disease activity in patients treated with teriflunomide
The specific study aims are to determine
1 the proportion of patients pre-treatment pre-Tx that have altered expression of one or more candidate markers in one or more T-subsets
2 whether expression levels of one or more of these markers in a T-subset changes on treatment On-Tx with teriflunomide
3 whether differences in the mRNA or protein kinetics of a putative marker predicts disease activity in patients treated with teriflunomide
4 whether expression levels of one or more target molecules Pre-Tx or On-Tx correlate with evidence of disease activity after one year on treatment
C STUDY DESIGN
C1 Design summary The study is a two-year prospective observational study of patients treated with teriflunomide The investigators will recruit up to 75 patients at baseline The investigators will study patients at baseline and at intervals On-Tx with teriflunomide in order to identify patients with active vs stable disease based on clinical relapse and radiological new T2 hyperintense lesions evidence The investigators will study the expression of biomarkers in each patient subgroup
C2 Primary endpoints The central question of the study is whether the expression levels of one or more putative biomarker TOB1 TLR2 TLR4 CCR1 differs between patients from the two informative subgroups
C3 Outcome measures The outcomes that will address the central question of the study consist of the following
1 Measures of protein expression levels of TLR2 TLR4 and CCR1 on CD4 T-subsets by flow cytometry
2 TOB1 mRNA expression in CD4 T-subsets by quantitative RT-PCR qRT-PCR and the PrimeFlow RNA Assay
3 Differences in mRNA or protein expression of selected markers in short-term cultures between patients from the two informative subgroups
C4 Subject population The study population includes
1 treatment-naive patients with RRMS from the Montreal Neurological Hospital MNH MS clinics before treatment pre-Tx with teriflunomide and
2 patients with RRMS that were previously treated with other disease modifiying therapies DMTs but that are switching to teriflunomide
C5 Ethics approval The study was approved by the Neurosciences Research Ethics Board of the MNHMN The data will be kept for 7 years
C6 Consents The Principal Investigator or his delegate will obtain informed consent
D STUDY PROCEDURES The study will investigate a biomarker signature in patients with MS who are being treated with teriflunomide by their neurologist
D1 Participants and clinic visits Before beginning treatment with teriflunomide each patient will have a clinical evaluation an MRI as part of routine clinical practice and a blood draw see below At intervals participants will have a clinical evaluation and a second MRI evaluation to determine whether they have active vs stable disease In order to permit a phased approach the neurologists will endeavor to identify patients after six months of treatment who show evidence of active vs stable disease approximately 3 in each subgroup Blood draws will be obtained from each of these patients for subsequent laboratory studies see below
D2 Sample collection Up to 120 ml of peripheral venous blood will be drawn from each subject at baseline and at intervals to collect serum and plasma to obtain total lymphocyte counts CD3 CD4 and CD8 counts and to isolate peripheral blood mononuclear cells PBMC for immediate analysis and also for cryopreservation
D3 Laboratory analysis
D31 Naïve CD4 T-cell isolation and quantitative RT-PCR analysis of TOB1 expression Reduced expression of TOB1 an inhibitor of T-cell proliferation correlated with rapid progression from CIS to RRMS in one study 2 and also correlated in the investigators previous study with rapid progression from RRMS to SPMS 2 The investigators will isolate naïve CD4 T-cells using MACS beads 2 and determine TOB1 expression by quantitative RT-PCR in a small cohort of patients 5-10 patients and age-matched healthy controls HCs
D32 Analysis of cryopreserved PBMC
Strategy 1 Surface protein expression of TLR2 TLR4 and CCR1 in CD4 T-subsets The investigators will analyze baseline and On-Tx cryopreserved samples for expression positivity and MFI of TLR2 TLR4 and CCR1 on naïve central memory effector memory terminally differentiated effector memory and regulatory T-cells by flow cytometry on a BD LSRFortessa with a 5-laser system The investigators will use FlowJo software for analysis The investigators will identify the biomarker which is most informative ie shows the greatest difference in expression levels between patients in the two informative subgroups at baseline or on treatment
Strategy 2 Comparison of TOB1 mRNA expression by qRT-PCR and the PrimeFlow RNA Assay The investigators will develop the PrimeFlow RNA assay affymetrix Ebioscience for simultaneous analysis of mRNA and protein expression Initially the investigators will compare TOB1 mRNA expression with that obtained by qRT-PCR as qRT-PCR is the gold standard for mRNA quantification The investigators will compare TOB1 mRNA expression in naïve CD4 T-cells by the two methods If the measurements are comparable the investigators will then be able to simultaneously assess TOB1 expression in naïve CD4 T-cells and the expression of the most informative biomarker in CD4 T-subsets and compare expression levels in patients from the two informative subgroups as outlined below
Strategy 3 mRNA and protein kinetics of TOB1 and a T-cell biomarker in stimulated T-cells The investigators anticipate that T-cell stimulation will identify differences between patients from the two informative subgroups in either mRNA or protein kinetics of TOB1 or one of our surface protein biomarkers In other words these studies may show differences between patients with active vs stable MS
This strategy involves a stimulation phase and an analytical phase as follows
1 Firstly after selecting patients from the two patient subgroups 3-4 patientssubgroup and from age-matched HCs the investigators will stimulate cryopreserved PBMC from baseline and one-year treatment samples using PMAionomycin and BD GolgiStop Protein Transport inhibitor for 4 hr at 37 C in 5 CO2 The investigators will determine the optimal concentrations of each reagent through assay-specific titration for use with the PrimeFlow RNA Assay
2 The second phase will use the PrimeFlow RNA Assay to assess mRNA and protein kinetics of TOB1 and the most informative surface protein biomarker in patient subgroups and HCs This assay can reveal the dynamics of both RNA and protein expression in individual cells and in T-subsets in response to T-cell stimulation The utility of the PrimeFlow RNA assay was validated in a landmark study of T-subsets 4 detailed methods are available on the following website httpwwwebiosciencecommedianewpdfPrimeFlowRNAAssayUM010915pdf
D4 Statistical analysis The investigators will use GraphPad Prism 7 for all statistical analyses and use appropriate parametric or non-parametric tests according to the data distribution
E ANTICIPATED RESULTS Firstly the investigators anticipate that one or more T-subsets in baseline PBMC samples before teriflunomide treatment will show increased surface protein expression of one or more T-cell activation molecules in the patients that show disease activity On-Tx vs patients that do not show disease activity On-Tx Such a finding may help identify patients for whom teriflunomide is the optimal DMT Secondly treatment with teriflunomide may alter the expression of one or more T-cell activation molecules in one or more T-subsets and this alteration may correlate with reduced disease activity thereby suggesting that altered T-cell activation contributes to a positive response to teriflunomide Thirdly short term cultures of baseline samples vs one-year samples from patients and from age-matched HCs may identify differences in mRNA and protein kinetics of a T-cell activation marker in patients at baseline which then revert to a normal pattern in those with no disease activity at one year Such findings would point towards important functional alterations mediated by teriflunomide that correlate with a positive therapeutic response
F STUDY SITES All patients will be recruited from the Montreal Neurological Hospital MS Clinics and all experimental work will be done in the Duff Medical Building Department of Pathology McGill University
G TIMELINES G1 First Patient In FPI - one month after execution of the contract and ethics approval
G2 Last Patient In LPI 12-15 months after execution of the contract and ethics approval this reflects the time required to recruit a total of up to 75 patients treated withTeriflunomide
G3 Interim analyses
Assess recruitment rate at 2 months to consider inclusion of satellite centers
RT-PCR analysis of TOB1 in 5-10 patients Pre-TX and age-matched HCs 4 months
Preliminary flow cytometry analysis of surface protein biomarkers based on the tentative identification of 3 active vs 3 stable patients identified at the 8 month On-Tx time point
Additional flow cytometry analysis of surface protein biomarkers on a further 3 active vs 3 stable patients identified at the 14 month On-Tx time point
G4 Progress Reports Quarterly progress updates on patient recruitment and biannual report on clinical evaluation parameters to subgroup patients
G5 Last Patient Visit LPLV - 24-27 months G6 Completion of Final Report - 30 months G7 Projected Manuscript Submission 33 months H APPENDICES APPENDIX 1 Primary Antibody Panel
1 FVS 510 BD
2 CD4 BUV395 BD
3 CD45RA BV421 BD
4 CD127 BUV737 BD
5 CD25 BV786 BD
6 CCR1 Alexa Fluor 488 RD Sys
7 TLR2 PE eBioscience
8 CCR7 PE-CF594 BD
9 CD14 PerCP-Cy55 BD
10 TLR4 APC eBioscience
11 CD3 APC-H7 BD FVS 510 BD Horizon Fixable Viability Stain 510 BUV BD Horizon Brilliant Ultraviolet BV BD Horizon Brilliant Violet Alexa Fluor Molecular Probes sometimes abbrev AF PE-CF594 BD Horizon PE-CF594 APC-H7 BD Pharmingen APPENDIX 2 PrimeFlow RNA Assay Panels including TLR2 as a possible biomarker - under development
A BACKGROUND Teriflunomide Aubagio is a once-daily oral immunomodulatory DMT for patients with relapsing-remitting MS RRMS 1 The objective of the present study is to determine whether a putative biomarker signature predicts disease activity in patients treated with teriflunomide The investigators previously identified a 130-gene signature associated with immune activation that identified patients with MS that had rapid transition to secondary progressive MS SPMS From this signature the investigators identified three genes TLR2 TLR4 and CCR1 which had increased protein expression on naïve CD4 T-cells in these patients The investigators also showed that mRNA for an anti-proliferation factor termed TOB1 was downregulated in these T-cells in patients with rapid MS progression The findings suggest therefore that naïve CD4 T-cell activation identifies patients with MS having a short RRMS duration 2
For this proposal the principal investigator suggests that various molecules involved in T-cell activation may also serve as useful biomarkers to predict treatment responses to teriflunomide
B STUDY OBJECTIVES
B1 Study objective and specific aims The objective is to determine whether the T-cell activation markers that were previously identified will predict disease activity in patients treated with teriflunomide
The specific study aims are to determine
1 the proportion of patients pre-treatment pre-Tx that have altered expression of one or more candidate markers in one or more T-subsets
2 whether expression levels of one or more of these markers in a T-subset changes on treatment On-Tx with teriflunomide
3 whether differences in the mRNA or protein kinetics of a putative marker predicts disease activity in patients treated with teriflunomide
4 whether expression levels of one or more target molecules Pre-Tx or On-Tx correlate with evidence of disease activity after one year on treatment
C STUDY DESIGN
C1 Design summary The study is a two-year prospective observational study of patients treated with teriflunomide The investigators will recruit up to 75 patients at baseline The investigators will study patients at baseline and at intervals On-Tx with teriflunomide in order to identify patients with active vs stable disease based on clinical relapse and radiological new T2 hyperintense lesions evidence The investigators will study the expression of biomarkers in each patient subgroup
C2 Primary endpoints The central question of the study is whether the expression levels of one or more putative biomarker TOB1 TLR2 TLR4 CCR1 differs between patients from the two informative subgroups
C3 Outcome measures The outcomes that will address the central question of the study consist of the following
1 Measures of protein expression levels of TLR2 TLR4 and CCR1 on CD4 T-subsets by flow cytometry
2 TOB1 mRNA expression in CD4 T-subsets by quantitative RT-PCR qRT-PCR and the PrimeFlow RNA Assay
3 Differences in mRNA or protein expression of selected markers in short-term cultures between patients from the two informative subgroups
C4 Subject population The study population includes
1 treatment-naive patients with RRMS from the Montreal Neurological Hospital MNH MS clinics before treatment pre-Tx with teriflunomide and
2 patients with RRMS that were previously treated with other disease modifiying therapies DMTs but that are switching to teriflunomide
C5 Ethics approval The study was approved by the Neurosciences Research Ethics Board of the MNHMN The data will be kept for 7 years
C6 Consents The Principal Investigator or his delegate will obtain informed consent
D STUDY PROCEDURES The study will investigate a biomarker signature in patients with MS who are being treated with teriflunomide by their neurologist
D1 Participants and clinic visits Before beginning treatment with teriflunomide each patient will have a clinical evaluation an MRI as part of routine clinical practice and a blood draw see below At intervals participants will have a clinical evaluation and a second MRI evaluation to determine whether they have active vs stable disease In order to permit a phased approach the neurologists will endeavor to identify patients after six months of treatment who show evidence of active vs stable disease approximately 3 in each subgroup Blood draws will be obtained from each of these patients for subsequent laboratory studies see below
D2 Sample collection Up to 120 ml of peripheral venous blood will be drawn from each subject at baseline and at intervals to collect serum and plasma to obtain total lymphocyte counts CD3 CD4 and CD8 counts and to isolate peripheral blood mononuclear cells PBMC for immediate analysis and also for cryopreservation
D3 Laboratory analysis
D31 Naïve CD4 T-cell isolation and quantitative RT-PCR analysis of TOB1 expression Reduced expression of TOB1 an inhibitor of T-cell proliferation correlated with rapid progression from CIS to RRMS in one study 2 and also correlated in the investigators previous study with rapid progression from RRMS to SPMS 2 The investigators will isolate naïve CD4 T-cells using MACS beads 2 and determine TOB1 expression by quantitative RT-PCR in a small cohort of patients 5-10 patients and age-matched healthy controls HCs
D32 Analysis of cryopreserved PBMC
Strategy 1 Surface protein expression of TLR2 TLR4 and CCR1 in CD4 T-subsets The investigators will analyze baseline and On-Tx cryopreserved samples for expression positivity and MFI of TLR2 TLR4 and CCR1 on naïve central memory effector memory terminally differentiated effector memory and regulatory T-cells by flow cytometry on a BD LSRFortessa with a 5-laser system The investigators will use FlowJo software for analysis The investigators will identify the biomarker which is most informative ie shows the greatest difference in expression levels between patients in the two informative subgroups at baseline or on treatment
Strategy 2 Comparison of TOB1 mRNA expression by qRT-PCR and the PrimeFlow RNA Assay The investigators will develop the PrimeFlow RNA assay affymetrix Ebioscience for simultaneous analysis of mRNA and protein expression Initially the investigators will compare TOB1 mRNA expression with that obtained by qRT-PCR as qRT-PCR is the gold standard for mRNA quantification The investigators will compare TOB1 mRNA expression in naïve CD4 T-cells by the two methods If the measurements are comparable the investigators will then be able to simultaneously assess TOB1 expression in naïve CD4 T-cells and the expression of the most informative biomarker in CD4 T-subsets and compare expression levels in patients from the two informative subgroups as outlined below
Strategy 3 mRNA and protein kinetics of TOB1 and a T-cell biomarker in stimulated T-cells The investigators anticipate that T-cell stimulation will identify differences between patients from the two informative subgroups in either mRNA or protein kinetics of TOB1 or one of our surface protein biomarkers In other words these studies may show differences between patients with active vs stable MS
This strategy involves a stimulation phase and an analytical phase as follows
1 Firstly after selecting patients from the two patient subgroups 3-4 patientssubgroup and from age-matched HCs the investigators will stimulate cryopreserved PBMC from baseline and one-year treatment samples using PMAionomycin and BD GolgiStop Protein Transport inhibitor for 4 hr at 37 C in 5 CO2 The investigators will determine the optimal concentrations of each reagent through assay-specific titration for use with the PrimeFlow RNA Assay
2 The second phase will use the PrimeFlow RNA Assay to assess mRNA and protein kinetics of TOB1 and the most informative surface protein biomarker in patient subgroups and HCs This assay can reveal the dynamics of both RNA and protein expression in individual cells and in T-subsets in response to T-cell stimulation The utility of the PrimeFlow RNA assay was validated in a landmark study of T-subsets 4 detailed methods are available on the following website httpwwwebiosciencecommedianewpdfPrimeFlowRNAAssayUM010915pdf
D4 Statistical analysis The investigators will use GraphPad Prism 7 for all statistical analyses and use appropriate parametric or non-parametric tests according to the data distribution
E ANTICIPATED RESULTS Firstly the investigators anticipate that one or more T-subsets in baseline PBMC samples before teriflunomide treatment will show increased surface protein expression of one or more T-cell activation molecules in the patients that show disease activity On-Tx vs patients that do not show disease activity On-Tx Such a finding may help identify patients for whom teriflunomide is the optimal DMT Secondly treatment with teriflunomide may alter the expression of one or more T-cell activation molecules in one or more T-subsets and this alteration may correlate with reduced disease activity thereby suggesting that altered T-cell activation contributes to a positive response to teriflunomide Thirdly short term cultures of baseline samples vs one-year samples from patients and from age-matched HCs may identify differences in mRNA and protein kinetics of a T-cell activation marker in patients at baseline which then revert to a normal pattern in those with no disease activity at one year Such findings would point towards important functional alterations mediated by teriflunomide that correlate with a positive therapeutic response
F STUDY SITES All patients will be recruited from the Montreal Neurological Hospital MS Clinics and all experimental work will be done in the Duff Medical Building Department of Pathology McGill University
G TIMELINES G1 First Patient In FPI - one month after execution of the contract and ethics approval
G2 Last Patient In LPI 12-15 months after execution of the contract and ethics approval this reflects the time required to recruit a total of up to 75 patients treated withTeriflunomide
G3 Interim analyses
Assess recruitment rate at 2 months to consider inclusion of satellite centers
RT-PCR analysis of TOB1 in 5-10 patients Pre-TX and age-matched HCs 4 months
Preliminary flow cytometry analysis of surface protein biomarkers based on the tentative identification of 3 active vs 3 stable patients identified at the 8 month On-Tx time point
Additional flow cytometry analysis of surface protein biomarkers on a further 3 active vs 3 stable patients identified at the 14 month On-Tx time point
G4 Progress Reports Quarterly progress updates on patient recruitment and biannual report on clinical evaluation parameters to subgroup patients
G5 Last Patient Visit LPLV - 24-27 months G6 Completion of Final Report - 30 months G7 Projected Manuscript Submission 33 months H APPENDICES APPENDIX 1 Primary Antibody Panel
1 FVS 510 BD
2 CD4 BUV395 BD
3 CD45RA BV421 BD
4 CD127 BUV737 BD
5 CD25 BV786 BD
6 CCR1 Alexa Fluor 488 RD Sys
7 TLR2 PE eBioscience
8 CCR7 PE-CF594 BD
9 CD14 PerCP-Cy55 BD
10 TLR4 APC eBioscience
11 CD3 APC-H7 BD FVS 510 BD Horizon Fixable Viability Stain 510 BUV BD Horizon Brilliant Ultraviolet BV BD Horizon Brilliant Violet Alexa Fluor Molecular Probes sometimes abbrev AF PE-CF594 BD Horizon PE-CF594 APC-H7 BD Pharmingen APPENDIX 2 PrimeFlow RNA Assay Panels including TLR2 as a possible biomarker - under development
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
True
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