Cerebrospinal Fluid-biomarkers-based Diagnostic and Prognostic Models for Multiple Sclerosis
Conditions: Multiple Sclerosis
Sponsor: Washington University School of Medicine

NCT04496830 
 
 
 
 
Cerebrospinal fluid-biomarkers-based diagnostic and prognostic models for  
multiple sclerosis  
 
 
 
 
Multi-center study: 
 
• Washington University in St. Louis (WUSTL): Dr. Anne H. Cross, Dr. Laura Piccio, 
Dr. Salim Chain, Dr. Laura Ghezzi 
• NIAID/NDS: Dr. Bibiana Bielekova, Dr. Peter Kosa 
• University of Colorado: Dr. Enrique Alvarez 
• University of Ottawa: Dr. Mark Freedman 
• University of Montana: Dr. Mark Greenwood 
 
 
 
 
Supported by: 
 
The National Multiple Sclerosis Society (NMSS-RG-1907-34348) 
 
 
 
 
 
 
 
 
 
 
Version 3 
April 28, 2022 
 
 

 
 
 
 
 
 
 
 
 
 
Cerebrospinal fluid-biomarkers-based diagnostic and prognostic models for  
multiple sclerosis  
 
 
 
 
Multi-center study: 
 
• Washington University in St. Louis (WUSTL): Dr. Anne H. Cross, Dr. Laura Piccio, 
Dr. Salim Chain, Dr. Laura Ghezzi 
• NIAID/NDS: Dr. Bibiana Bielekova, Dr. Peter Kosa 
• University of Colorado: Dr. Enrique Alvarez 
• University of Ottawa: Dr. Mark Freedman 
• University of Montana: Dr. Mark Greenwood 
 
 
 
 
Supported by: 
 
The National Multiple Sclerosis Society (NMSS-RG-1907-34348) 
 
 
 
 
 
 
 
 
 
 
Version 3 
April 28, 2022 

TABLE OF CONTENTS 
Page 
 
SYNOPSIS ………..………………………………………………………………………………. 
 
1. 
STUDY DESIGN ……………………………………………………………...………….. 
 
2.         SELECTION AND ENROLLMENT OF SUBJECTS ……………………...…………. 
 
 
2.1 
Eligibility criteria………………………………………………………..……….. 
 
2.2 
Study enrollment procedures: collection of data/samples at follow up visits ……. 
 
2.3 
Targeted patient number for each SPINCOMS center ……………….……… 
 
5.         STUDY INTERVENTIONS ………………………………………………….…………. 
 
 
5.1 
Interventions, Administration, and Duration ...……………………………….……….. 
 
5.2 
Handling of Study Interventions ……………………………………………….……… 
 
5.3 
Concomitant Interventions ……………………………………………………….……. 
 
5.4 
Adherence Assessment ………………………………………………………………... 
 
6.         CLINICAL AND LABORATORY EVALUATIONS …………………………………. 
 
 
6.1 
Schedule of Evaluations …………………………………………………….…………. 
 
6.2 
Timing of Evaluations ………………………………………………………….……… 
 
6.3 
Special Instructions and Definitions of Evaluations ………………………….……….. 
 
 
 
7.          MANAGEMENT OF ADVERSE EXPERIENCES ……...……………………...……. 
 
8.          CRITERIA FOR INTERVENTION DISCONTINUATION ………………...………. 
 
9.          STATISTICAL CONSIDERATIONS …………………………………………...…….. 
 
 
9.1 
General Design Issues ………………………………………………………..………... 
 
9.2 
Outcomes ……………………………………………………………………...………. 
 
9.3 
Sample Size and Accrual ……………………………………………………..……….. 
 
9.4 
Data Monitoring ………………………………………………………………..……... 
 
9.5 
Data Analyses …………………………………………………………………...…….. 
 
10.          DATA COLLECTION, SITE MONITORING, AND ADVERSE EXPERIENCE    
               REPORTING …………………………………………………………………………... 
 
 
10.1 
Records to be Kept ………………………………………………………………..…… 
 
10.2 
Role of Data Management ………………………………………………………..…… 
 
10.3 
Quality Assurance …………………………………………………………………...… 
 
10.4 
Adverse Experience Reporting ……………………………………………………..…. 

 
11.          HUMAN SUBJECTS ……………………………………………………………..……. 
 
 
11.1 
Institutional Review Board (IRB) Review and Informed Consent ………………...….. 
 
11.2 
Subject Confidentiality …………………………………………………………..……. 
 
11.3 
Study Modification/Discontinuation ………………………………………….…..…… 
 
12.          PUBLICATION OF RESEARCH FINDINGS ………………………………...…….. 
 
13.          REFERENCES ………………………………………………………………...……….. 
 
 
APPENDICES 
 
MODEL INFORMED CONSENT 
(typical appendix items are outcome scales, flowcharts, and organizational charts) 
 
 

SYNOPSIS 
 
Study title 
Cerebrospinal fluid-biomarkers-based diagnostic and prognostic models for multiple sclerosis  
  
Summary and Objectives 
Using machine learning, we have developed from SOMAScan:  
1. A molecular diagnostic test that differentiates MS from other inflammatory and non-
inflammatory central nervous system (CNS) diseases (area under receiver-operator characteristic 
curve-AUROC of 0.98);  
2. A molecular test that differentiates relapsing-remitting MS from progressive MS variants 
(AUROC of 0.91); and  
3. A molecular test that predicts future rates of disability progression, concordance coefficient of 
0.425 (p<0.001).  
 
Because these results are derived from a single research center (NIAID/NDS), it is imperative to 
determine their performance in real clinical practice settings as a necessary step for their potential 
regulatory approval. Consequently, this application has 2 specific aims:  
 
AIM 1. To independently validate afore-mentioned CSF-biomarker-based tests for their clinical value 
within the multicenter Spinal fluid Consortium for MS (SPINCOMS). In Aim 1, each of the 3 defined 
tests will be validated in 100 new SPINCOMS patients. To validate the prognostic test, 100 MS patients 
with CSF collected at least 3 years from the date of a follow-up study visit will be evaluated at follow-
up examination with standardized clinical outcomes. CSF will be analyzed blinded using pre-defined 
statistical models. 
 
AIM 2. To explore whether collected CSF-biomarkers point towards pathogenic heterogeneity that may 
predict patient-specific efficacy for different disease-modifying treatments (DMTs) or identify 
pathogenic mechanisms not targeted by current DMTs. In Aim 2, clustering analysis will assess 
pathogenic heterogeneity and explore potential predictors of response to therapy. 
 
1.  STUDY DESIGN 
Cross-sectional cohort studies (Figure 1.). 
1. For the MS diagnostic test, we will select 50 patients with definite MS (CIS/RIS excluded) and 
50 controls, including 25 non-inflammatory neurological diseases (NIND) that mimic MS (such 
as patients with vascular-ischemic changes, complex migraines, leukodystrophies, etc.) and 25 
other inflammatory neurological diseases (OIND) such as NMO, Susac’s syndrome, sarcoidosis, 
CNS vasculitis. 
2. For validating the RRMS versus progressive MS classifier, we will select CSF from 50 RRMS 
and 50 progressive MS patients (25 SPMS and 25 PPMS).  

3. Cryopreserved CSF samples from these patients will be analyzed blindly using SOMAscan 
assay. Probability of MS diagnosis and progressive MS diagnosis will be calculated based on 
published models2. Results will then be unblinded and analyzed for accuracy of diagnostic 
conclusion.  
4. To evaluate the MS severity score, we will select 50 RRMS and 50 progressive MS (25 PPMS 
and 25 SPMS).  
 
 
 
The cohorts of MS patients in 1-3 can overlap. 
Select n=100-130 eligible MS patients 
Required: ≥ 50 RRMS, ≥ 25 PPMS, ≥ 25 SPMS 
 
2.  SELECTION AND ENROLLMENT OF SUBJECTS 
2.1 Eligibility criteria 
MS Patients selection criteria (n=100; Table 1) 
- 
Lumbar puncture (LP) in the untreated stage with cryopreserved CSF (serum/blood optional) 
with consent to use it for future research 
- 
≥ 3 years of follow-up from LP (with respect to the date of the scheduled follow up study visit) 
- 
At time of LP untreated and not treated with steroid or off steroids ≥ one month 
- 
Available/willing to come for in-person follow-up 
- 
Available/willing to sign the NIH 09-I-0032 “Sample processing only” consent form 
- 
Diagnosis of MS based on 2017 McDonald criteria at time of follow-up visit 
 
Non-MS Patients selection criteria (n=50) 
Required: 25 NIND, 25 OIND  
- 
Lumbar puncture (LP) in the untreated stage with cryopreserved CSF (serum/blood optional) 
with consent to use it for future research 
- 
≥ 3 years of follow-up from LP 
- 
At time of LP untreated and not treated with steroid or off steroids ≥ one month 
- 
Up to date contact information  
- 
Available/willing to sign the NIH 09-I-0032 “Sample processing only” consent form 
- 
Diagnosis: 
NIND: e.g., ischemic-gliotic changes, CADASIL and other leukodystrophies, migraines, 
ischemic spinal cord lesions etc 
1. MS diagnostic classifier
2. RRMS vs PMS classifier
3. MS severity model
50 MS, 50 non-MS 
non-MS diagnoses: ~25 NIND & ~25 
OIND with high diagnostic certainty 
(CIS/RIS must be excluded, unless 
they progressed to definite MS)
50 RRMS, 50 PMS 
PMS: ~25 PPMS & ~25 SPMS
Can overlap w Cohort 1 &3
50 RRMS, 50 PMS 
PMS: ~25 PPMS & ~25 SPMS
Minimum 3 years from untreated LP
Can overlap w Cohort 1 &2
Figure 1. 

OIND: e.g. CNS Sjogren’s, SLE, vasculitis, CNS infections, MOG-associated disorders, NMO 
spectrum disorders (NMOSD) 
 
Data and procedure at follow-up visit for MS patients: 
- 
Signed consent for the present study and procedures. 
• Will include acknowledgment of NIH sample processing consent. 
- 
Collection of a blood sample: one serum tube, four sodium heparin tubes and two potassium 
EDTA tubes [or one PAXGene tube] (see Figure 2 at the end of the protocol). 
- 
Vital signs, weight, height and BMI. 
- 
Complete neurological examination documented in NeurEx (recorded with an iPAD). 
- 
Complete clinical data questionnaire with the patient. 
- 
25FW & non-dominant hand 9HPT (required for calculating CombiWISE & MS-DSS). 
- 
Smartphone Apps (include 25FW, SDMT and tests that correlate highly w 9HPT – can be 
acquired in patient-autonomous manner with minimal assistance). 
- 
Optical Coherence Tomography (OCT). 
- 
Propose optional LP to be done at the NIH. 
 
Data and procedure at phone interview visit for MS patients: 
- 
Phone interview consent for the present study and procedures. 
• Will include acknowledgment of NIH sample processing consent. 
- 
Complete clinical data questionnaire with the patient. 
- 
Propose optional LP to be done at the NIH. 
 
Additional data to be collected after consent is obtained: 
MS Patient data collected after follow-up or phone interview visit 
Access electronic medical record (EMR) to collect/confirm the following information: 
- 
Transcribing documented neurological examination completed within 1 month of the time of LP 
to EDSS or NeurEx. 
- 
Detailed DMT history (with estimated date of initiation and termination of all drugs). 
- 
Documentation of proximity of clinical relapse or contrast enhancing lesions (CELs) to the initial 
LP. 
- 
Estimation of subsequent relapse rate; conversion to clinically-definite MS (for original 
RIS/CIS) and possibly SPMS (for original RRMS). 
- 
Imaging Data 
- If available, Brain MRI ± 3 month of the LP date: 
• If images available transfer de-identified MRI images 
• If images are not available collect the following information: 
1) CEL if MRI done within a month from LP 
2) T2 lesion load (for MRI done within 3 months from LP) 
3) T1 black holes  
- If available, spinal cord MRI ± 3 months of LP date:  
• If images available transfer de-identified MRI images  
• If images are not available collect the following information: 
1) CEL if MRI done within a month from LP 
2) T2 lesion load (for MRI done within 3 months from LP) 
      - If available, Brain MRI (within 1 year of follow-up visit): 

• If images available transfer de-identified MRI images 
• If images are not available collect the following information: 
1) CEL if MRI done within a month from the follow-up visit 
2) T2 lesion load  
3) T1 black holes  
- Spinal cord MRI around the time of follow-up visit: 
• 
If images available transfer de-identified MRI images 
• 
If images are not available collect the following information: 
1) CEL if MRI done within a month from follow-up visit 
2) T2 lesion load 
 
- 
Available information for calculating BREMSO score (i.e., symptom onset: sphincter, motor, 
sensory; number of neurological functional systems involved at onset; incomplete recovery at 
onset). 
- 
If available, 25FW/9HPT around the time of initial LP. 
 
MS Patient data collected under consent waiver 
Access electronic medical record (EMR) to collect/confirm the following information: 
- 
Transcribing documented neurological examination completed within 1 month of the time of LP 
to EDSS or NeurEx. 
- 
Transcribing documented most recently completed neurological examination available in 
medical record to EDSS or NeurEx. 
- 
Detailed DMT history (with estimated date of initiation and termination of all drugs). 
- 
Documentation of proximity of clinical relapse or contrast enhancing lesions (CELs) to the initial 
LP. 
- 
Estimation of subsequent relapse rate; conversion to clinically-definite MS (for original 
RIS/CIS) and possibly SPMS (for original RRMS). 
- 
Available information for calculating BREMSO score (i.e., symptom onset: sphincter, motor, 
sensory; number of neurological functional systems involved at onset; incomplete recovery at 
onset). 
- 
If available, 25FW/9HPT around the time of initial LP. 
 
 
Non-MS Patient data collected after follow-up visit: 
- 
Access electronic medical record (EMR) to confirm the clinical diagnosis. 
- 
Contact the patient on the phone to confirm their availability to be contacted by the NIH to sign 
their consent. 
 
 
 
 
 
 
 

2.3   Targeted patient number for each SPINCOMS center. 
Table 1. 
MS patients
All require in-person follow-up visit
Non-MS patients
Do not require in-person follow-up 
but require data collection
Wash. U
60 (30 RRMS, 30PMS)
30 (~15 NIND, ~15 OIND)
U. Ottawa
20 (10 RRMS, 10PMS)
10 (~5 NIND, ~5 OIND)
U. Colorado
20 (10 RRMS, 10PMS)
10 (~5 NIND, ~5 OIND)
NIAID/NDS
None budgeted, but will contribute all 
available that are non-overlapping and 
unpublished
None budgeted, but will contribute all 
available that are non-overlapping and 
unpublished
 
 
*University of Montana will not participate in patient recruitment, data analysis only 
 
2.4 Study identification numbers and data entry for enrolled subjects across centers 
 
Each patient will be assigned sequential alphanumeric code and generation of all CSF biomarker data 
will be performed under this code, by laboratory personnel having no access to any clinical or imaging 
data. 
 
Study identifiers for the clinical data will be assigned by REDCap as follows: assigned 3-digit Site ID 
(###) then 3-digit patient ID (001, 002, etc.). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3.  SUMMARY OF CLINICAL AND LABORATORY EVALUATIONS 
 
Table 2. List of procedures 
Assessment 
Follow up 
visit 
Phone 
Interview 
Under Waiver 
of Consent 
Data from time 
of LP  
(≥ 3 before 
follow up) 
Informed Consent 
X 
X 
 
 
Check availability to be 
contacted by NIH and to sign 
NIAID/NDS consent for 
“Sample processing only”  
X 
X 
 
 
Vital signs; weight/height and 
BMI 
X 
* 
* 
* 
Complete the patient 
questionnaire 
X 
X 
 
 
Access EMR to obtain 
complete clinical data (disease 
history, personal and familial 
history of autoimmune 
diseases, detailed DMT history) 
X 
X 
X 
 
Access EMR to obtain imaging 
data (MRI images obtained 
around the time of LP and from 
the most recent MRI will be 
used) 
 
X  
 
 
X 
CSF 
 
 
 
X 
(cryopreserved 
and available) 
Check availability to repeat LP 
at NIH 
X 
 
 
 
Blood 
X 
 
 
* 
NeuroEx 
X 
 
 
* 
25FW/9HPT 
X 
 
 
* 
SDMT 
X 
 
 
* 
OCT 
X 
 
 
* 
Smartphone tests 
X 
 
 
 
X=mandatory for the study 
* = collected if data available 
 
 
 
 

3.1 Special Instructions and Definitions of Evaluations 
 
Informed Consent 
The potential participant and one of the study team members will review the consent together. 
The potential participant will be given time to read through the consent, ask questions and consult 
with relatives. The PI or the study coordinator will be responsible for the consent process. They 
will explain the study consent form in detail, and they will be available for any questions potential 
study participants may have.  
 
To participate in this study, patients will also need to sign a separate consent for “sample 
processing only” by NDS/NIAID that includes genotyping of common SNPs using microarrays – 
this is NOT whole exome or whole genome sequencing. This will be a separate consent, supplied 
by NIH to research patients for review and once signed, returned directly to NIH through mail 
using FedEx. 
 
Disease history and treatment history 
Information will be obtained from the patient’s clinical medical record and from the patient 
questionnaire. All information related to this study will be kept in an individual study binder for 
each patient. Enrollment in the study (as well as a possible premature end of the study visit) will 
be recorded in their clinical charts.  
 
Clinical and Laboratory Assessments 
 
Vital Signs: weight/height (BMI), blood pressure and heart rate 
 
Blood sample collection: 7 tubes of blood (approximately 2.5 tablespoons; Figure 2). 
 
Neurological examination/NeuroEx: NeuroEx is a standardized and digitalized neurological 
examination that automatically calculates most common clinician-generated MS (and related 
diseases) disability scales. The NeurEx App is developed on FileMaker Pro platform and 
available in clinics on mobile platforms such as iPad (Kosa et al., 2018). 
 
Cognitive Testing: standardized testing to thinking, memory, hand/arm coordination, and 
walking ability (Timed 25-FT Walk, 9-Hole Peg Test & Symbol Digit Modality Test (SDMT). 
 
Optical Coherence Tomography (OCT): non-invasive photographing of the retina that will 
measure the nerve layer and thickness. 
 
Smartphone tests: smartphone apps, employing smart-phone embedded sensors, have been 
developed by NIH to assess disease-related disability in a patient-autonomous manner. A series of 
11 cognitive and motor tests have been implemented to obtain outcomes that better correlate with 
clinical-assessed disability. The tests will be taken autonomously by the patients in about 30 
minutes (Boukhvalova et al., 2019; Boukhvalova et al., 2018).  
 
          Laboratory Studies 
At follow-up visit we will collect blood that will be used for research purposes.  

These are the procedures that are planned: 
• 
Serum collection: serum will be aliquoted and stored at -80°C  
• 
Peripheral blood mononuclear cell isolation to freeze down aliquots in freezing media 
(5x106 cells/aliquot) to be stored in liquid nitrogen for future analyses. 
• 
 K2 EDTA purple top tube or PAXgene Blood DNA tube for collection of genomic DNA 
 
4. DATA COLLECTION 
Records to Be Kept 
 
Indicate what information will be retained for each subject and by whom.  Describe methods for 
maintaining confidentiality of subject records. 
Paper/hard copy records (hard copy surveys, questionnaires, case report forms, pictures, etc.). 
Patient data will be maintained in individual study binders. All binders will be maintained in a 
locked file cabinet in the locked office of the coordinator in a locked suite. 
Electronic records (computer files, electronic databases, etc.) - The data will be compiled into a 
password protected Excel file, where the patients will only be identified by their coded study ID. 
This file will be stripped of identifiers prior to sending to the statistician for analysis. The master 
list will be kept in separate password protected file that only the study coordinator and PI will 
have access to. This list will be destroyed at final analysis. The data will be housed on secure 
server. Electronic data will also be entered into REDCAP database. This database is password 
protected.  
 
5. HUMAN SUBJECTS 
Institutional Review Board (IRB) Review and Informed Consent 
 
This protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB or ethics committee responsible for oversight of the study at 
each site.  A signed consent form will be obtained from the subject. For subjects who cannot 
consent for themselves, such as those below the legal age, a parent, legal guardian, or person 
with power of attorney, must sign the consent form; additionally, the subject's assent must also 
be obtained if he or she is able to understand the nature, significance, and risks associated with 
the study.  The consent form will describe the purpose of the study, the procedures to be 
followed, and the risks and benefits of participation.  A copy of the consent form will be given to 
the subject, parent, or legal guardian, and this fact will be documented in the subject’s record. 
Subject Confidentiality 
 
All laboratory specimens, evaluation forms, reports, and other records that leave the site will be 
identified only by the Study Identification Number (SID) to maintain subject confidentiality.  All 
records will be kept in a locked file cabinet.  All computer entry and networking programs will 

be done using SIDs only.  Clinical information will not be released without written permission of 
the subject, except as necessary for monitoring by IRB, the FDA, the NIAID, the OHRP, the 
sponsor, or the sponsor’s designee. 
Study Modification/Discontinuation 
 
The study may be modified or discontinued at any time by the IRB, the NIAID the sponsor, the 
OHRP, the FDA, or other government agencies as part of their duties to ensure that research 
subjects are protected. 
 
 
PUBLICATION OF RESEARCH FINDINGS 
Results of this trial will be made available to the public.  
 
REFERENCES 
Kosa P, Barbour C, Wichman A, Sandford M, Greenwood M, Bielekova B. NeurEx: digitalized 
neurological examination offers a novel high-resolution disability scale. Annals of clinical and 
translational neurology 2018 
 
Boukhvalova AK, Kowalczyk E, Harris T, Kosa P, Wichman A, Sandford MA, Memon A, 
Bielekova B. dentifying and Quantifying Neurological Disability via Smartphone. Front Neurol. 
2018 Sep 4;9:740. doi: 10.3389/fneur.2018.00740. 
 
Boukhvalova AK, Fan O, Weideman AM, Harris T, Kowalczyk E, Pham L, Kosa P, Bielekova 
B. Smartphone Level Test Measures Disability in Several Neurological Domains for Patients 
With Multiple Sclerosis. Front Neurol. 2019 May 28;10:358. doi: 10.3389/fneur.2019.00358 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 2. Blood collection tubes. 
 
 
PROCEDURE FOR BLOOD PROCESSING 
 
FREEZING MEDIA:  75% FBS, 15% RPMI, 10% DMSO 
Isolation of PBMCs from whole blood: CONFIRM that the consent for SPINCOM study was 
collected!! 
1. Pour the blood in a 50ml falcon, dilute the sample with an equal volume of PBS. Mix gently. 
2. In another 50 ml Falcon add 12-15 ml of Ficoll, add the blood on top of it, really slowly, in order 
to form two layers. 
3. Centrifuge at 3000 RPM for 30 minutes at room temperature, with the brake off. 
4. Draw up the ring with a Pasteur and transfer it in a new 50ml falcon. 
5. Wash with PBS (full falcon) for two times, centrifuge at 1750 RPM, for 5 minutes at 4°C and 
decant the supernatant. Resuspend the pellet. 
6. Add 10 to 20 ml of PBS and resuspend well the pellet in it. Count the cells: 
• 
If < 15x106 cells Freeze at least 2 aliquots of PBMCs 
• 
If at least 15x106 cells -> take 5x106 cells and transfer in a 15ml tube – for Trizol  
• 
If > 15x106 cells proceed to do more aliquots of frozen PBMCs 
7. Wash one more time with PBS, centrifuge at 1200 RPM, for 5 minutes at 4°C and decant the 
supernatant. Resuspend the pellet. (In the 15 ml tube add 700ul of Trizol. Transfer in a 1.5 ml 
Eppendorf and store in a -80°C freezer). 
8. To freeze the PBMCs add freezing media to the pellet (per cryovial: 5x106 cells in 1ml). 
9. Place the vials in the Cryofreezing container and place it in a -80°C freezer. 
 
➢ On the Eppendorf and cryovials write: patient ID, PBMC numbers, date.