Biomarkers and Disease Activity in Patients Treated With Teriflunomide (Aubagio)
Condition: Multiple Sclerosis, Relapsing-Remitting
MeSH terms: Multiple Sclerosis; Multiple Sclerosis, Relapsing-Remitting
Study type: Observational

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 2(TLR2), 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 change(s) 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) differ(s) 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
      MNH/MN. 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.

      D3.1. 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).

      D3.2. 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, i.e. 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 patients/subgroup)
           and from age-matched HCs, the investigators will stimulate cryopreserved PBMC from
           baseline and one-year treatment samples using PMA/ionomycin 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:
           http://www.ebioscience.com/media/newpdf/PrimeFlowRNAAssayUM010915.pdf.

      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 G.1. First Patient In (FPI) - one month after execution of the contract and
      ethics approval.

      G.2. 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.

      G.3. 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 (R&D Sys.)

        7. TLR2 PE (eBioscience)

        8. CCR7 PE-CF594 (BD)

        9. CD14 PerCP-Cy5.5 (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
    

Intervention name(s): Teriflunomide
Intervention type(s): Drug
Intervention description(s): Each patient will receive 1 tablet (14 mg) on a daily basis.
Other intervention name(s): Aubagio
Intervention MeSH terms: Teriflunomide
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Patients with relapsing-remitting MS who agree to be treated with teriflunomide
      
Biospecimen description: 
      Cryopreserved peripheral blood mononuclear cells (PBMC)
    
Keywords: biomarker; teriflunomide; activity