Clinical Validation of a Multi-protein, Serum-based Assay for Disease Activity Assessments in Multiple Sclerosis

Background and objectives An unmet need exists for validated quantitative tools to measure multiple sclerosis (MS) disease activity and progression. We developed a custom immunoassay-based MS disease activity (MSDA) Test incorporating 18 protein concentrations into an algorithm to calculate four Disease Pathway scores (Immunomodulation, Neuroinflammation, Myelin Biology, and Neuroaxonal Integrity) and an overall Disease Activity score. The objective was to clinically validate the MSDA Test based on associations between scores and clinical/radiographic assessments. Methods Serum samples (N=614) from patients with MS at multiple sites were split into Train (n=426; algorithm development) and Test (n=188; evaluation) subsets. Subsets were stratified by demographics, sample counts per site, and gadolinium-positive (Gd+) lesion counts; age and sex were used to demographically adjust protein concentrations. MSDA Test results were evaluated for potential association with Gd+ lesion presence/absence, new and enlarging (N/E) T2 lesion presence, and active versus stable disease status (composite endpoint combining radiographic and clinical evidence of disease activity). Results A multi-protein model was developed (trained and cross-validated) using the Train subset. When applied to the Test subset, the model classified the Gd+ lesion presence/absence, N/E T2 lesion presence, and active versus stable disease status assessments with an area under the receiver operating characteristic (AUROC) of 0.781, 0.750, and 0.768, respectively. In each case, the multi-protein model had significantly (bootstrapped, one-sided p <0.05) greater AUROC performance when compared with the top-performing, demographically adjusted (by age and sex) single-protein model based on neurofilament light polypeptide chain. Algorithmic score thresholds corresponded to low, moderate, or high levels of disease activity. Based on the Test subset, the diagnostic odds ratios determined that the odds of having ≥1 Gd+ lesions among samples with a moderate/high Disease Activity score were 4.49 times that of a low Disease Activity score. The odds of having ≥2 Gd+ lesions among samples with a high Disease Activity score were 20.99 times that of a low/moderate Disease Activity score. Discussion The MSDA Test was clinically validated; the multi-protein model had greater performance compared with the top-performing single-protein model. The MSDA Test may serve as a quantitative and objective tool to enhance care for MS. ### Competing Interest Statement Tanuja Chitnis has received compensation for consulting from Biogen, Novartis Pharmaceuticals, Roche Genentech, and Sanofi Genzyme, and has received research support from the National Institutes of Health, National MS Society, US Department of Defense, EMD Serono, I-Mab Biopharma, Mallinckrodt ARD, Novartis Pharmaceuticals, Octave Bioscience, Inc., Roche Genentech, and Tiziana Life Sciences. This research was conducted in part with the support of the Department of Defense through the Multiple Sclerosis Research Program under Award No. W81XWH-18-1-0648 (to T. Chitnis). John Foley has received research support from Biogen, Novartis, Adamas, Octave Bioscience, Inc., Genentech, and Mallinckrodt, has received speakers' honoraria and acted as a consultant for EMD Serono, Genzyme, Novartis, Biogen, and Genentech, has equity interest in Octave Bioscience Inc., and is the founder of InterPro Bioscience. Carolina Ionete has received research support from Biogen, Serono, Genentech, NMSS, and Department of Defense, and received compensation for advisory board activity from Sanofi-Genzyme. Nabil K. El Ayoubi has received support to attend scientific educational courses from Novartis, Merck Serono, Sanofi, Biologix, and has received speaker honoraria for scientific presentations on Multiple Sclerosis from Biologix, Sanofi, Merck Serono, and Novartis. Shrishti Saxena, Patricia Gaitan-Walsh, Anu Paul, and Fermisk Saleh have no disclosures. Hrishikesh Lokhande has received research support from the US Department of Defense and Octave Bioscience, Inc. Howard Weiner has received research support from the Department of Defense, Genentech, Inc., National Institutes of Health, National Multiple Sclerosis Society, Novartis and Sanofi Genzyme. He has received compensation for consulting from Genentech, Inc, IM Therapeutics, IMAB Biopharma, MedDay Pharmaceuticals, Tiziana Life Sciences and vTv Therapeutics. Jennifer L. Venzie is an employee of The Lockwood Group and provided editorial support funded by Octave Bioscience, Inc. Ferhan Qureshi, Anisha Keshavan, Kian Jalaleddini, and Ati Ghoreyshi are employees of Octave Bioscience, Inc. Michael J. Becich, Fatima Rubio da Costa, Victor M. Gehman, and Fujun Zhang were employees of Octave Bioscience, Inc., at the time the study was completed. Samia J. Khoury has received compensation for scientific advisory board activity from Merck and Roche. ### Funding Statement This study was funded by Octave Bioscience, Inc. and in part by the U.S. Department of Defense (W81XWH2110633 to T Chitnis). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Mass General Brigham institutional review board (Somerville, MA, USA) and the WCG institutional review board (Puyallup, WA, USA). All patients provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Access to anonymized data not published within this article and the study protocol can be made available by request from any qualified investigator once a data-sharing agreement is in place. * AMIR : American University of Beirut Medical Center Study ANOVA : analysis of variance AUROC : area under the receiver operating characteristic CD6 : cluster of differentiation 6 CIS : clinically isolated syndrome CLIMB : Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Women’s Hospital CNS : central nervous system CSF : cerebral spinal fluid CXCL13 : C-X-C motif chemokine ligand 13 DMT : disease-modifying therapy FSDD : Family Study of Demyelinating Disease Gd+ : gadolinium-positive IL-12β : interleukin 12β LOQ : limit of quantitation MRI : magnetic resonance imaging MS : multiple sclerosis MSDA : multiple sclerosis disease activity N/E : new and enlarging NfL : neurofilament light polypeptide chain NPV : negative predictive value NPX : normalized protein expression OLS : ordinary least squares PPV : positive predictive value RMMSC : Rocky Mountain Multiple Sclerosis Clinic RRMS : relapsing-remitting multiple sclerosis SUMMIT : Serially Unified Multicenter Multiple Sclerosis Investigation TNFSF13B : tumor necrosis factor superfamily member 13B