Biomarkers for predicting multiple sclerosis disease progression

Abstract

A method for early detection and monitoring of MS disease progression by analyzing expression levels of biomarkers alone or in combination with MRI volumetric measurements from a sample obtained from a subject.SOLUTION: A method for analyzing quantitative expression values of biomarkers of a biomarker panel for determining multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) in a human subject. Further provided are kits for measuring quantitative expression values of markers, as well as computer systems and software for a predictive model for determining multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) in a human subject based on the quantitative expression values of the markers.SELECTED DRAWING: FIG. 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 074,768, filed September 4, 2020, and U.S. Provisional Patent Application No. 63 / 148,906, filed February 12, 2021, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes. Summary of the Invention

[0002] Generally, MRI scans are routinely performed to confirm MS disease progression in subjects. However, MRI scans are expensive and time-consuming to perform. Measuring a patient's MS status more frequently allows for more agile clinical management. Disclosed herein are methods for predicting multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) using a multivariate biomarker panel that analyzes the quantitative expression levels of biomarkers in samples obtained from a subject. Samples, such as samples obtained by blood sampling, are simpler, faster, and less expensive than MRI. Therefore, analyzing the expression levels of biomarkers in samples obtained from a subject, either in combination with MRI volumetrics or by themselves, allows for early detection and monitoring of MS disease progression.

[0003] Further disclosed herein is a non-transitory computer-readable medium for predicting multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) using a multivariate biomarker panel. Further disclosed herein is a kit comprising a set of reagents for determining expression levels of multivariate biomarkers that are useful for predicting multiple sclerosis disease activity (e.g., multiple sclerosis disease progression). Further disclosed herein is a system for predicting multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) using a multivariate biomarker panel.

[0004] Advantages of multivariate biomarker panels for detecting multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) include: • Improved sensitivity: Multi-biomarker tests improve performance (area under the curve (AUC, also referred to herein as AUROC)), precision), especially by eliminating false negatives that cannot be detected by individual biomarkers. • Detection of silent progression: Multi-biomarker testing can detect asymptomatic progression, which manifests as radiographic atrophy but not as worsening symptoms. Specificity: Individual biomarkers are often differentially expressed in other neurological conditions. Multi-biomarker testing may help distinguish disease progression specific to multiple sclerosis. Predictive power: Multivariate models incorporating shifts in biomarker levels identify patients progressing toward progressive increase or decrease in active disease (with stronger performance than individual biomarkers alone).

[0005] Disclosed herein is a method for predicting multiple sclerosis disease progression in a subject, the method comprising: obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, the plurality of biomarkers comprises one or more biomarkers from at least one group selected from Group 1, Group 2, and Group 3; Group 1 includes one or more of biomarker 1, biomarker 2, biomarker 3, biomarker 4, biomarker 5, biomarker 6, biomarker 7, and biomarker 8; Biomarker 1 is GFAP, NEFL, OPN, CXCL9, MOG, or CHI3L1; biomarker 2 is CDCP1, IL-18BP, IL-18, GFAP, or MSR1; biomarker 3 is MOG, CADM3, KLK6, BCAN, OMG, or GFAP; biomarker 4 is CXCL13, NOS3, or MMP-2; biomarker 5 is OPG, TFF3, or ENPP2; biomarker 6 is APLP1, SEZ6L, BCAN, DPP6, NCAN, or KLK6; biomarker 7 is VCAN, TINAGL1, CANT1, NECTIN2, MMP-9, or NPDC1; and biomarker 8 is NEFL, MOG, CADM3, or GFAP. Group 2 includes one or more of biomarker 9, biomarker 10, biomarker 11, biomarker 12, biomarker 13, biomarker 14, biomarker 15, biomarker 16, and biomarker 17; biomarker 9 is CXCL9, CXCL10, IL-12B, CXCL11, or GFAP; biomarker 10 is TNFRSF10A, TNFRSF11A, SPON2, CHI3L1, or IFI30; biomarker 11 is CCL20, CCL3, or TWEAK; biomarker 12 is TNFSF13B, CXCL16, ALCAM, or IL-18; biomarker 13 is OPN, OMD, MEPE, or GFAP; biomarker 14 is SERPINA9, TNFRSF9, or CNTN4; biomarker 15 is CD6, CD5, CRTAM, CD244, or TNFRSF9; biomarker 16 is FLRT2, DDR1, NTRK2, CDH6, MMP-2; and biomarker 17 is CNTN2, DPP6, GDNFR-α-3, or SCARF2. Group 3 includes biomarker 18, biomarker 19, biomarker 20, and biomarker 21; Biomarker 18 is COL4A1, IL-6, Notch3, or PCDH17; biomarker 19 is GH, GH2, or IGFBP-1; biomarker 20 is IL-12B, IL12A, or CXCL9; and biomarker 21 is PRTG, NTRK2, NTRK3, or CNTN4. And, Generating predictions of multiple sclerosis disease activity by applying a predictive model to the expression levels of multiple biomarkers Includes:

[0006] In various embodiments, the plurality of biomarkers includes each biomarker in Group 1, where biomarker 1 is GFAP, biomarker 2 is CDCP1, biomarker 3 is MOG, biomarker 4 is CXCL13, biomarker 5 is OPG, biomarker 6 is APLP1, biomarker 7 is VCAN, and biomarker 8 is NEFL. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.31. 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.77. In various embodiments, the performance of the predictive model is characterized by a PPV of 0.19.

[0007] In various embodiments, the plurality of biomarkers further comprises each biomarker in Group 2, wherein biomarker 9 is CXCL9, biomarker 10 is TNFRSF10A, biomarker 11 is CCL20, biomarker 12 is TNFSF13B, biomarker 13 is OPN, biomarker 14 is SERPINA9, biomarker 15 is CD6, biomarker 16 is FLRT, and biomarker 17 is CNTN2. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2) In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.76. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.19.

[0008] In various embodiments, the plurality of biomarkers further includes each biomarker in Group 3, wherein biomarker 18 is COL4A1, biomarker 19 is GH, biomarker 20 is IL-12B, and biomarker 21 is PRTG. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.36. 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.19.

[0009] In various embodiments, the plurality of biomarkers includes one or more biomarkers of Group 1, wherein the one or more biomarkers of Group 1 include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72-0.78. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0010] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient between 0.015 and 0.090. 2 It is characterized by coefficients.

[0011] In various embodiments, the prediction of multiple sclerosis disease progression is a measure of brain parenchymal fraction. In various embodiments, the prediction of multiple sclerosis disease progression is a measure of Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6 or greater indicates severe MS disease progression. In various embodiments, the prediction of multiple sclerosis disease progression is a measure of Patient-Determined Disease Stage (PDDS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression. In various embodiments, the prediction of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0012] Further disclosed herein is a method for predicting multiple sclerosis disease progression in a subject, the method comprising: one or more axonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immunomodulatory biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; or one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN. obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, including at least one of: Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers Further disclosed herein is a method for predicting multiple sclerosis disease progression in a subject, the method comprising: one or more axonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immunomodulatory biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN; one or more cerebrovascular function biomarkers selected from the group consisting of COL4A1, VCAN, GFAP, and CD6. obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, including at least one of: Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers Includes:

[0013] In various embodiments, the one or more axonal integrity biomarkers include NEFL, OPG, and GFAP, the one or more neuroinflammatory biomarkers include CXCL13 and CXCL9, the one or more immunomodulatory biomarkers include CDCP1, and the one or more myelination biomarkers include MOG and APLP1. In various embodiments, the one or more axonal integrity biomarkers further include SERPINA9, FLRT2, and CNTN2, the one or more neuroinflammatory biomarkers further include CCL20, CXCL9, TNFRSF10A, and CD6, the one or more immunomodulatory biomarkers further include TNFSF13B, and the one or more myelination biomarkers further include OPN. In various embodiments, the one or more axonal integrity biomarkers further include PRTG, and the one or more immunomodulatory biomarkers further include IL-12B.

[0014] In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.17.

[0015] In various embodiments, the plurality of biomarkers includes one or more axonal integrity biomarkers, wherein the one or more axonal integrity biomarkers include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72-0.78. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0016] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R of 0.015 to 0.090. 2 It is characterized by coefficients.

[0017] In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value. In various embodiments, the predictor of multiple sclerosis disease progression is an Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6.0 or greater indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0018] Further disclosed herein is a method for predicting multiple sclerosis disease progression in a subject, the method comprising: obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, the plurality of biomarkers comprising two or more of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG; Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers In various embodiments, the plurality of biomarkers comprises each of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG.

[0019] In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.17.

[0020] In various embodiments, the plurality of biomarkers includes GFAP. In various embodiments, the plurality of biomarkers further includes CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes CXCL13. In various embodiments, the plurality of biomarkers further includes MOG. In various embodiments, the plurality of biomarkers further includes OPG. In various embodiments, the plurality of biomarkers further includes CDCP1 and APLP1. In various embodiments, the plurality of biomarkers further includes MOG and CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1 and CXCL13. In various embodiments, the plurality of biomarkers further includes CDCP1 and SERPINA9. In various embodiments, the plurality of biomarkers further includes MOG and CXCL13. In various embodiments, the plurality of biomarkers further includes CDCP1, CCL20, and APLP1. In various embodiments, the plurality of biomarkers further includes CDCP1, APLP1, and CXCL13. In various embodiments, the plurality of biomarkers further comprises CDCP1, CCL20, and MOG. In various embodiments, the plurality of biomarkers further comprises CDCP1, APLP1, and SERPINA9. In various embodiments, the plurality of biomarkers further comprises CDCP1, MOG, and APLP1. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72 to 0.78.

[0021] In various embodiments, the plurality of biomarkers further includes MOG. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes OPG. In various embodiments, the plurality of biomarkers further includes TNFRSF10A. In various embodiments, the plurality of biomarkers further includes CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes NEFL. In various embodiments, the plurality of biomarkers further includes CNTN2. In various embodiments, the plurality of biomarkers further includes GH. In various embodiments, the plurality of biomarkers further includes CXCL9. In various embodiments, the plurality of biomarkers further includes OPG and MOG. In various embodiments, the plurality of biomarkers further includes OPG and APLP1. In various embodiments, the plurality of biomarkers further includes TNFRSF10A and MOG. In various embodiments, the plurality of biomarkers further includes CXCL9 and OPG. In various embodiments, the plurality of biomarkers further comprises TNFRSF10A and APLP1. In various embodiments, the plurality of biomarkers further comprises APLP1 and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL13 and APLP1. In various embodiments, the plurality of biomarkers further comprises FLRT2 and APLP1. In various embodiments, the plurality of biomarkers further comprises CXCL9 and APLP1. In various embodiments, the plurality of biomarkers further comprises GH and APLP1. In various embodiments, the plurality of biomarkers further comprises CXCL9, OPG, and MOG. In various embodiments, the plurality of biomarkers further comprises CNTN2, OPG, and MOG. In various embodiments, the plurality of biomarkers further comprises CXCL9, OPG, and APLP1. In various embodiments, the plurality of biomarkers further comprises OPG, PRTG, and MOG. In various embodiments, the plurality of biomarkers further comprises OPG, OPN, and MOG.In various embodiments, the plurality of biomarkers further comprises CXCL13, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises FLRT2, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises OPN, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL9, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL13, FLRT2, and APLP1. In various embodiments, the performance of the predictive model has a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0022] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the plurality of biomarkers includes CDCP1 and OPG. In various embodiments, the plurality of biomarkers includes CDCP1 and SERPINA9. In various embodiments, the plurality of biomarkers includes OPG and TNFRSF10A. In various embodiments, the plurality of biomarkers includes OPG and MOG. In various embodiments, the plurality of biomarkers includes CDCP1 and MOG. In various embodiments, the plurality of biomarkers includes CDCP1, MOG, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, SERPIN A9, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, OPG, and CXCL13. In various embodiments, the plurality of biomarkers includes CDCP1, CXCL9, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, FLRT2, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, MOG, OPG, and CXCL13. In various embodiments, the plurality of biomarkers comprises CDCP1, MOG, TNFRSF10A, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, CXCL9, SERPINA9, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, CNTN2, SERPINA9, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, SERPINA9, CD6, and OPG. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70.

[0023] In various embodiments, the plurality of biomarkers includes OPG and NEFL. In various embodiments, the plurality of biomarkers includes OPG and OPN. In various embodiments, the plurality of biomarkers includes OPG and FLRT2. In various embodiments, the plurality of biomarkers includes OPG and MOG. In various embodiments, the plurality of biomarkers includes CXCL9 and OPG. In various embodiments, the plurality of biomarkers includes GH and NEFL. In various embodiments, the plurality of biomarkers includes CXCL13 and NEFL. In various embodiments, the plurality of biomarkers includes APLP1 and NEFL. In various embodiments, the plurality of biomarkers includes CCL20 and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9 and NEFL. In various embodiments, the plurality of biomarkers includes OPG, MOG, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, FLRT2, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9, OPG, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, CDCP1, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, OPN, and NEFL. In various embodiments, the plurality of biomarkers includes GH, APLP1, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CDCP1, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL13, CCL20, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CCL20, and NEFL. In various embodiments, the plurality of biomarkers includes CDCP1, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers includes CD6, CXCL9, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL.In various embodiments, the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and MOG. In various embodiments, the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and MOG. In various embodiments, the performance of the predictive model has a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient between 0.015 and 0.090. 2 It is characterized by coefficients.

[0024] In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value. In various embodiments, the predictor of multiple sclerosis disease progression is an Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of 6 or less indicates mild / moderate MS disease progression, and an EDSS score above 6.5 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score above 4 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a Multiple Sclerosis Rating Scale-Revised (MSRS-R) score. In various embodiments, generating a prediction of multiple sclerosis disease progression by applying a predictive model to expression levels of the plurality of biomarkers further includes applying the predictive model to one or more subject attributes, where the subject attributes include any of age, sex, and disease duration. In various embodiments, generating a prediction of multiple sclerosis disease progression includes comparing the score output by the predictive model to a reference score. In various embodiments, the reference score corresponds to any of the following: A) EDSS score; B) brain parenchymal fraction; C) PDDS score; D) PROMIS score; or E) MSRS-R score. In various embodiments, the reference score further corresponds to mild / moderate MS disease progression or severe MS disease progression. In various embodiments, the expression levels of the plurality of biomarkers are determined from a test sample obtained from the subject. In various embodiments, the test sample is a blood sample or a serum sample. In various embodiments, the subject has, is suspected of having, or has previously been diagnosed with multiple sclerosis.In various embodiments, obtaining or having obtained the dataset comprises performing an immunoassay to determine expression levels of a plurality of biomarkers. In various embodiments, the immunoassay is a proximity extension assay (PEA) or a LUMINEX xMAP multiplex assay. In various embodiments, performing the immunoassay comprises contacting the test sample with a plurality of reagents including antibodies. In various embodiments, the antibodies comprise one of monoclonal and polyclonal antibodies. In various embodiments, the antibodies comprise both monoclonal and polyclonal antibodies. In various embodiments, the methods disclosed herein further comprise selecting a therapy to administer to the subject based on the prediction of multiple sclerosis disease progression. In various embodiments, the methods disclosed herein further comprise determining the therapeutic efficacy of a therapy previously administered to the subject based on the prediction of multiple sclerosis disease activity. In various embodiments, determining the therapeutic efficacy of a therapy comprises comparing the prediction to a previous prediction determined for the subject at a previous time point. In various embodiments, determining the therapeutic efficacy of a therapy comprises determining that the therapy is effective depending on the difference between the prediction and the previous prediction. In various embodiments, determining the therapeutic efficacy of a therapy comprises determining that the therapy lacks efficacy in response to a lack of difference between the prediction and the previous prediction.

[0025] Further disclosed herein is a non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: obtaining a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises one or more biomarkers of at least one group selected from Group 1, Group 2, and Group 3, wherein Group 1 comprises one or more of biomarker 1, biomarker 2, biomarker 3, biomarker 4, biomarker 5, biomarker 6, biomarker 7, and biomarker 8, wherein biomarker 1 is GFAP, NEFL, OPN, CXCL9, MOG, or CHI3L1; biomarker 2 is CDCP1, IL-18BP, IL-18, GFAP, or MSR1; biomarker 3 is MOG, CADM3, KLK6, BCAN, OMG, or GFAP; and biomarker 4 is CXCL13,biomarker 5 is OPG, TFF3, or ENPP2; biomarker 6 is APLP1, SEZ6L, BCAN, DPP6, NCAN, or KLK6; biomarker 7 is VCAN, TINAGL1, CANT1, NECTIN2, MMP-9, or NPDC1; and biomarker 8 is NEFL, MOG, CADM3, or GFAP, where Group 2 is biomarker 9, biomarker 10, biomarker 11 , biomarker 12, biomarker 13, biomarker 14, biomarker 15, biomarker 16, and biomarker 17, wherein biomarker 9 is CXCL9, CXCL10, IL-12B, CXCL11, or GFAP, biomarker 10 is TNFRSF10A, TNFRSF11A, SPON2, CHI3L1, or IFI30, biomarker 11 is CCL20, CCL3, or TWEAK, and biomarker 12 is TNF biomarker 15 is CD6, CD5, CRTAM, CD244, or TNFRSF9; biomarker 16 is FLRT2, DDR1, NTRK2, CDH6, MMP-2; and biomarker 17 is CNTN2, DPP6, GDNFR-α-3, or SCARF2, Group 3 includes one or more of biomarker 18, biomarker 19, biomarker 20, and biomarker 21, wherein biomarker 18 is COL4A1, IL-6, Notch3, or PCDH17, biomarker 19 is GH, GH2, or IGFBP-1, biomarker 20 is IL-12B, IL12A, or CXCL9, and biomarker 21 is PRTG, NTRK2, NTRK3, or CNTN4; Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers An order to perform Includes:

[0026] In various embodiments, the plurality of biomarkers includes each biomarker in Group 1, where biomarker 1 is GFAP, biomarker 2 is CDCP1, biomarker 3 is MOG, biomarker 4 is CXCL13, biomarker 5 is OPG, biomarker 6 is APLP1, biomarker 7 is VCAN, and biomarker 8 is NEFL. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.31. 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.77. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.19.

[0027] In various embodiments, the plurality of biomarkers further comprises each biomarker in Group 2, wherein biomarker 9 is CXCL9, biomarker 10 is TNFRSF10A, biomarker 11 is CCL20, biomarker 12 is TNFSF13B, biomarker 13 is OPN, biomarker 14 is SERPINA9, biomarker 15 is CD6, biomarker 16 is FLRT, and biomarker 17 is CNTN2. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.76. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.19.

[0028] In various embodiments, the plurality of biomarkers further includes each biomarker in Group 3, wherein biomarker 18 is COL4A1, biomarker 19 is GH, biomarker 20 is IL-12B, and biomarker 21 is PRTG. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.36. 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.19.

[0029] In various embodiments, the plurality of biomarkers includes one or more biomarkers of Group 1, wherein the one or more biomarkers of Group 1 include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72-0.78. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0030] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient between 0.015 and 0.090. 2In various embodiments, the prediction of multiple sclerosis disease progression is a measure of brain parenchymal fraction. In various embodiments, the prediction of multiple sclerosis disease progression is a measure of Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6 or greater indicates severe MS disease progression. In various embodiments, the prediction of multiple sclerosis disease progression is a measure of Patient-Determined Disease Stage (PDDS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the predictor of multiple sclerosis disease progression is a Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0031] Further disclosed herein is a non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: one or more axonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immunomodulatory biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN. obtaining a dataset comprising expression levels of a plurality of biomarkers, including at least one of: Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers An order to perform Further disclosed herein is a non-transitory computer readable medium for determining multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: one or more axonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immunomodulatory biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN; or one or more cerebrovascular function biomarkers selected from the group consisting of COL4A1, VCAN, GFAP, and CD6. obtaining a dataset comprising expression levels of a plurality of biomarkers, including at least one of: Generating predictions of multiple sclerosis disease progression by applying a predictive model to the expression levels of multiple biomarkers An order to perform Includes:

[0032] In various embodiments, the one or more axonal integrity biomarkers include NEFL, OPG, and GFAP, the one or more neuroinflammatory biomarkers include CXCL13 and CXCL9, the one or more immunomodulatory biomarkers include CDCP1, and the one or more myelination biomarkers include MOG and APLP1. In various embodiments, the one or more axonal integrity biomarkers further include SERPINA9, FLRT2, and CNTN2, the one or more neuroinflammatory biomarkers further include CCL20, CXCL9, TNFRSF10A, and CD6, the one or more immunomodulatory biomarkers further include TNFSF13B, and the one or more myelination biomarkers further include OPN. In various embodiments, the one or more axonal integrity biomarkers further include PRTG, and the one or more immunomodulatory biomarkers further include IL-12B.

[0033] In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 ). In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.17. In various embodiments, the plurality of biomarkers includes one or more axon integrity biomarkers, the one or more axon integrity biomarkers including GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72-0.78. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0034] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70. In various embodiments, the performance of the predictive model is characterized by a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient between 0.015 and 0.090. 2 In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value. In various embodiments, the predictor of multiple sclerosis disease progression is an Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6.0 or greater indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of more than 4 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0035] Further disclosed herein is a non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: obtaining a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises two or more of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG; Applying a predictive model to the expression levels of multiple biomarkers to generate a prediction of multiple sclerosis disease progression. An order to perform In various embodiments, the plurality of biomarkers comprises each of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG.

[0036] In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 ) In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.74. In various embodiments, the performance of the predictive model is characterized by a PPV of at least 0.17.

[0037] In various embodiments, the plurality of biomarkers includes GFAP. In various embodiments, the plurality of biomarkers further includes CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes CXCL13. In various embodiments, the plurality of biomarkers further includes MOG. In various embodiments, the plurality of biomarkers further includes OPG. In various embodiments, the plurality of biomarkers further includes CDCP1 and APLP1. In various embodiments, the plurality of biomarkers further includes MOG and CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1 and CXCL13. In various embodiments, the plurality of biomarkers further includes CDCP1 and SERPINA9. In various embodiments, the plurality of biomarkers further includes MOG and CXCL13. In various embodiments, the plurality of biomarkers further includes CDCP1, CCL20, and APLP1. In various embodiments, the plurality of biomarkers further includes CDCP1, APLP1, and CXCL13. In various embodiments, the plurality of biomarkers further comprises CDCP1, CCL20, and MOG. In various embodiments, the plurality of biomarkers further comprises CDCP1, APLP1, and SERPINA9. In various embodiments, the plurality of biomarkers further comprises CDCP1, MOG, and APLP1. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.70. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.72 to 0.78.

[0038] In various embodiments, the plurality of biomarkers further includes MOG. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes OPG. In various embodiments, the plurality of biomarkers further includes TNFRSF10A. In various embodiments, the plurality of biomarkers further includes CDCP1. In various embodiments, the plurality of biomarkers further includes APLP1. In various embodiments, the plurality of biomarkers further includes NEFL. In various embodiments, the plurality of biomarkers further includes CNTN2. In various embodiments, the plurality of biomarkers further includes GH. In various embodiments, the plurality of biomarkers further includes CXCL9. In various embodiments, the plurality of biomarkers further includes OPG and MOG. In various embodiments, the plurality of biomarkers further includes OPG and APLP1. In various embodiments, the plurality of biomarkers further includes TNFRSF10A and MOG. In various embodiments, the plurality of biomarkers further includes CXCL9 and OPG. In various embodiments, the plurality of biomarkers further comprises TNFRSF10A and APLP1. In various embodiments, the plurality of biomarkers further comprises APLP1 and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL13 and APLP1. In various embodiments, the plurality of biomarkers further comprises FLRT2 and APLP1. In various embodiments, the plurality of biomarkers further comprises CXCL9 and APLP1. In various embodiments, the plurality of biomarkers further comprises GH and APLP1. In various embodiments, the plurality of biomarkers further comprises CXCL9, OPG, and MOG. In various embodiments, the plurality of biomarkers further comprises CNTN2, OPG, and MOG. In various embodiments, the plurality of biomarkers further comprises CXCL9, OPG, and APLP1. In various embodiments, the plurality of biomarkers further comprises OPG, PRTG, and MOG. In various embodiments, the plurality of biomarkers further comprises OPG, OPN, and MOG.In various embodiments, the plurality of biomarkers further comprises CXCL13, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises FLRT2, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises OPN, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL9, APLP1, and NEFL. In various embodiments, the plurality of biomarkers further comprises CXCL13, FLRT2, and APLP1. In various embodiments, the performance of the predictive model has a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient of 0.11 to 0.22. 2 It is characterized by coefficients.

[0039] In various embodiments, the plurality of biomarkers does not include GFAP. In various embodiments, the plurality of biomarkers includes CDCP1 and OPG. In various embodiments, the plurality of biomarkers includes CDCP1 and SERPINA9. In various embodiments, the plurality of biomarkers includes OPG and TNFRSF10A. In various embodiments, the plurality of biomarkers includes OPG and MOG. In various embodiments, the plurality of biomarkers includes CDCP1 and MOG. In various embodiments, the plurality of biomarkers includes CDCP1, MOG, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, SERPIN A9, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, OPG, and CXCL13. In various embodiments, the plurality of biomarkers includes CDCP1, CXCL9, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, FLRT2, and OPG. In various embodiments, the plurality of biomarkers includes CDCP1, MOG, OPG, and CXCL13. In various embodiments, the plurality of biomarkers comprises CDCP1, MOG, TNFRSF10A, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, CXCL9, SERPINA9, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, CNTN2, SERPINA9, and OPG. In various embodiments, the plurality of biomarkers comprises CDCP1, SERPINA9, CD6, and OPG. In various embodiments, the performance of the predictive model is characterized by an AUROC of at least 0.65. In various embodiments, the performance of the predictive model is characterized by an AUROC of 0.66-0.70.

[0040] In various embodiments, the plurality of biomarkers includes OPG and NEFL. In various embodiments, the plurality of biomarkers includes OPG and OPN. In various embodiments, the plurality of biomarkers includes OPG and FLRT2. In various embodiments, the plurality of biomarkers includes OPG and MOG. In various embodiments, the plurality of biomarkers includes CXCL9 and OPG. In various embodiments, the plurality of biomarkers includes GH and NEFL. In various embodiments, the plurality of biomarkers includes CXCL13 and NEFL. In various embodiments, the plurality of biomarkers includes APLP1 and NEFL. In various embodiments, the plurality of biomarkers includes CCL20 and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9 and NEFL. In various embodiments, the plurality of biomarkers includes OPG, MOG, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, FLRT2, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9, OPG, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, CDCP1, and NEFL. In various embodiments, the plurality of biomarkers includes OPG, OPN, and NEFL. In various embodiments, the plurality of biomarkers includes GH, APLP1, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CDCP1, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL13, CCL20, and NEFL. In various embodiments, the plurality of biomarkers includes GH, CCL20, and NEFL. In various embodiments, the plurality of biomarkers includes CDCP1, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers includes CD6, CXCL9, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers includes CXCL9, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers includes CD6, CDCP1, CXCL13, and NEFL.In various embodiments, the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and MOG. In various embodiments, the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and NEFL. In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and MOG. In various embodiments, the performance of the predictive model has a Pearson's R of at least 0.10. 2 In various embodiments, the performance of the predictive model is characterized by a Pearson's R coefficient between 0.015 and 0.090. 2 In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value. In various embodiments, the predictor of multiple sclerosis disease progression is an Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of 6 or less indicates mild / moderate MS disease progression, and an EDSS score above 6.5 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score. In various embodiments, the predictor of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score above 4 indicates severe MS disease progression. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0041] In various embodiments, generating a prediction of multiple sclerosis disease progression by applying the predictive model to expression levels of the plurality of biomarkers further includes applying the predictive model to one or more subject attributes of the subject, where the subject attributes include any of age, sex, and disease duration. In various embodiments, generating a prediction of multiple sclerosis disease progression includes comparing the score output by the predictive model to a reference score. In various embodiments, the reference score corresponds to any of the following: A) EDSS score; B) brain parenchymal fraction value; C) PDDS score; D) PROMIS score; or E) MSRS-R score. In various embodiments, the reference score further corresponds to mild / moderate MS disease progression or severe MS disease progression.

[0042] In various embodiments, the expression levels of the plurality of biomarkers are determined from a test sample obtained from the subject. In various embodiments, the test sample is a blood or serum sample. In various embodiments, the subject has, is suspected of having, or has previously been diagnosed with multiple sclerosis. In various embodiments, obtaining or obtaining the dataset comprises performing an immunoassay to determine the expression levels of the plurality of biomarkers. In various embodiments, the immunoassay is a proximity extension assay (PEA) or a LUMINEX xMAP multiplex assay. In various embodiments, performing the immunoassay comprises contacting the test sample with a plurality of reagents comprising an antibody. In various embodiments, the antibody comprises one of a monoclonal antibody and a polyclonal antibody. In various embodiments, the antibody comprises both a monoclonal antibody and a polyclonal antibody. In various embodiments, the non-transitory computer-readable medium comprises: instructions that, when executed by a processor, cause the processor to select a therapy for administration to a subject based on a prediction of multiple sclerosis disease progression. In various embodiments, the non-transitory computer readable medium further comprises: instructions that, when executed by a processor, cause the processor to determine the therapeutic efficacy of a therapy previously administered to a subject based on the prediction of multiple sclerosis disease progression. In various embodiments, the instructions that cause the processor to determine the therapeutic efficacy of the therapy further include: instructions that, when executed by a processor, cause the processor to compare this prediction with a previous prediction determined for the subject at a previous time. In various embodiments, the instructions that cause the processor to determine the therapeutic efficacy of the therapy further include: instructions that, when executed by a processor, cause the processor to determine that the treatment is indicative of efficacy as a function of a difference between the prediction and a previous prediction; In various embodiments, the instructions that cause the processor to determine the therapeutic efficacy of the therapy further include: instructions that, when executed by a processor, cause the processor to determine that the treatment is lacking in efficacy in response to a lack of difference between the prediction and a prior prediction. Further includes:

[0043] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief explanation of the drawings]

[0044] [Figure 1A] 1 shows an overview of an environment for assessing a subject's disease progression via a disease progression prediction system, according to one embodiment. [Figure 1B] FIG. 1 is an exemplary block diagram of a disease progression system, according to an embodiment. [Figure 1C] 1 illustrates an exemplary set of training data, according to an embodiment. [Figure 1D] 1 shows exemplary biomarkers and their classifications. [Figure 2A] 1 shows the sequential forward selection of features using the sample from the F6 study. [Figure 2B] 1 shows the sequential forward selection of features using the sample from the F4 study. [Figure 3A] 1 shows the ROC curves of the multivariate model (training and testing) compared to the univariate model using neurofilament light as the single feature. [Figure 3B] Shows the confusion matrix for the multivariate model. [Figure 4A] Figure 1 shows the sequential forward selection of biomarkers for the cross-sectional classification of the presence / absence of radiologically defined disease activity. [Figure 4B] 1 shows ROC curves of trained models for predicting disease activity (minimal, normal, and extreme disease activity). [Figure 4C] Confusion matrices for the minimal disease model, the normal disease model, and the extreme disease model are shown. [Figure 4D] 1 shows the sequential progressive selection of biomarkers to predict disease severity according to predicted lesion counts. [Figure 5A] 1 illustrates the sequential forward selection of features to build a model for predicting annual relapse rate (ARR). [Figure 5B] 1 shows the ROC curve of the trained model for predicting annual recurrence rate as high (≧0.8) or low (<0.3). [Figure 6A] 1 shows the sequential forward selection of features to build a model for classifying worsening versus quiescent disease state in two separate patient cohorts. [Figure 6B] 1 shows the ROC curve of a trained model for predicting clinically defined disease as worsening versus quiescent. [Figure 7] We present a sequential, forward selection of features on absolute quantification data from the Expanded Disability Status Scale (EDSS). [Figure 8] 1A, 1B, and 1C illustrate an exemplary computer for executing the entities shown in FIGS. [Figure 9] Univariate analysis of individual biomarkers for predicting brain parenchymal fraction is shown. [Figure 10] 1 shows a multivariate analysis of combinations of individual biomarkers for predicting brain parenchymal fraction. [Figure 11] 1 shows multivariate analysis of biomarker combinations for predicting brain parenchymal fraction quartiles. [Figure 12] Associations of biomarkers (NfL and GFAP) with gender, disease duration, and race / ethnicity are shown. [Figure 13A] Characterization of patient-reported outcomes (Patient-Determined Disease Stage (PDDS), Patient-Reported Outcomes Measurement Information System (PROMIS), and Multiple Sclerosis Rating Scale-Revised (MSRS-R)) is presented. [Figure 13B] Characterization of patient-reported outcomes (Patient-Determined Disease Stage (PDDS), Patient-Reported Outcomes Measurement Information System (PROMIS), and Multiple Sclerosis Rating Scale-Revised (MSRS-R)) is presented. [Figure 13C] Characterization of patient-reported outcomes (Patient-Determined Disease Stage (PDDS), Patient-Reported Outcomes Measurement Information System (PROMIS), and Multiple Sclerosis Rating Scale-Revised (MSRS-R)) is presented. [Figure 14A-1] Correlation matrices revealing the association between individual biomarkers and PDDS, PROMIS, and MSRS-R are shown. [Figure 14A-2] See the description of Figure 14A-1. [Figure 14A-3] See the description of Figure 14A-1. [Figure 14A-4] See the description of Figure 14A-1. [Figure 14A-5] See the description of Figure 14A-1. [Figure 14B] Univariate analysis of individual biomarkers for predicting PDDS outcome is shown. [Figure 14C] Quantile-quantile plots of expected versus observed p-values ​​for severity of disability are shown. [Figure 14D] Classification of PDDS-defined severity (e.g., mild / moderate vs. severe) by univariate protein analysis (NfL, CD6, and CXCL13) is shown. [Figure 15A]1 shows classification of PDDS-defined severity (e.g., mild / moderate vs. severe) by multivariate biomarker analysis (NfL, CD6, and CXCL13). [Figure 15B] Receiver operating characteristic (ROC) curves and precision-recall curves for classifying PDDS-defined severity (e.g., mild / moderate vs. severe) through multivariate biomarker analysis (NfL, CD6, and CXCL13) are shown. [Figure 16] Univariate analyses of individual biomarkers for predicting PROMIS or MSRS-R outcomes are shown. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description I. Definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0046] The term "subject" includes human or non-human cells, tissues, or organisms, whether male or female, in vivo, ex vivo, or in vitro.

[0047] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0048] The term "sample" can include an aliquot of a bodily fluid, such as a single cell or multiple cells or cell fragments or a blood sample, obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention, or other means known in the art. Examples of aliquots of bodily fluids include amniotic fluid, aqueous humor, bile, lymph, milk, interstitial fluid, blood, plasma, earwax, Cowper's fluid, chyle, chyme, female vaginal fluid, menses, mucus, saliva, urine, vomit, tears, vaginal fluid, sweat, serum, semen, serum, sebum, pus, pleural fluid, cerebrospinal fluid, synovial fluid, intracellular fluid, and vitreous humor.

[0049] The term "disease activity" encompasses disease activity of any neurodegenerative disease, including multiple sclerosis, Parkinson's disease, Lewy body disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, Huntington's disease, spinal muscular atrophy, Friedreich's ataxia, and Batten disease.

[0050] The term "multiple sclerosis" or "MS" encompasses all forms of multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), progressive relapsing multiple sclerosis (PRMS), and clinically isolated syndrome (CIS).

[0051] As used herein, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to a diagnosis of multiple sclerosis (MS), the presence or absence of MS (e.g., usual disease, minimal disease), a shift in disease activity (e.g., increasing or decreasing), disease progression, MS severity, relapses or inflammatory events associated with MS, future or impending relapses or inflammatory events, relapse rate (e.g., annual relapse rate), MS status (e.g., worsening or quiescent), confirmation of no evidence of disease, the response of a subject diagnosed with multiple sclerosis to therapy, It refers to any measurable that provides information on the degree of disability from multiple sclerosis, the risk (e.g., likelihood) that a subject will subsequently develop multiple sclerosis, the change in multiple sclerosis compared to previous measurements (e.g., a patient's longitudinal change relative to baseline measurements), disease activity, or the differential diagnosis of types of multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), progressive relapsing multiple sclerosis (PRMS), and clinically isolated syndrome (CIS).

[0052] In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to a diagnosis of multiple sclerosis (MS). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the presence or absence of MS (e.g., regular disease, minimal disease). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to shifts in disease activity (e.g., increases or decreases). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the severity of MS. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to relapses or inflammatory events associated with MS. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to future or impending relapses or inflammatory events. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the relapse rate (e.g., annual relapse rate). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the MS status (e.g., worsening or quiescence). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the confirmation of no evidence of the disease status. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the response of a subject diagnosed with multiple sclerosis to a therapy. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the degree of disability from multiple sclerosis. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the risk (e.g., likelihood) of a subject subsequently developing multiple sclerosis. In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to changes in multiple sclerosis compared to previous measurements (e.g., longitudinal changes in a patient relative to a baseline measurement). In one embodiment, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to measurables that provide information on disease activity.In some embodiments, the terms "multiple sclerosis disease activity" or "multiple sclerosis disease activity" do not include the progression of MS (e.g., MS disease progression). Specifically, in such embodiments disclosed herein, the biomarker panel used to predict "multiple sclerosis disease activity" is different from the biomarker panel used to predict "multiple sclerosis disease progression."

[0053] In various embodiments, measurables informative of MS disease activity include measures of minimal disease activity (e.g., the presence or absence of a specific number of gadolinium-enhancing lesions, e.g., exactly one lesion), typical disease activity (e.g., the presence or absence of one or more gadolinium-enhancing lesions), a shift in disease activity (e.g., the appearance or disappearance of an active gadolinium-enhancing lesion), or the severity of disease activity (e.g., a larger number of gadolinium-enhancing lesions, where more gadolinium-enhancing lesions indicate increased disease severity). In one embodiment, measures informative of MS disease activity include measures of minimal disease activity (e.g., the presence or absence of a specific number of gadolinium-enhancing lesions, e.g., exactly one lesion). In one embodiment, measures informative of MS disease activity include measures of typical disease activity (e.g., the presence or absence of one or more gadolinium-enhancing lesions). In one embodiment, measures informative of MS disease activity include measures of a shift in disease activity (e.g., the appearance or disappearance of an active gadolinium-enhancing lesion). In one embodiment, the metrics informative of MS disease activity include metrics of disease activity severity (e.g., a higher number of gadolinium-enhancing lesions, with more gadolinium-enhancing lesions indicating greater disease severity).

[0054] In certain embodiments, the term "multiple sclerosis disease activity" or "multiple sclerosis disease activity" refers to the progression of MS (e.g., MS disease progression). In one embodiment, the measures that inform MS disease activity include measures of disease progression. Examples of measures of disease progression include the Expanded Disability Status Scale (EDSS), brain parenchymal fraction (BPF), atrophy measured by brain volume loss, or volumetric measurements of specific anatomical brain regions. Additional measures of disease progression may include patient-reported outcome measures such as the Patient-Determined Disease Stage (PDDS), PRO Measurement Information System (PROMIS), Multiple Sclerosis Rating Scale-Revised (MSRS-R), timed 25-foot walk (T25-FW), or hand / arm function measured by the 9-Hole Peg Test (9-HPT).

[0055] In various embodiments, MS disease progression refers to progressing to a milestone of MS disability, such as mild MS, moderate MS, or severe MS. Thus, the measure of MS disease progression may correspond to progression to one or more of mild MS, moderate MS, or severe MS. For example, for the MS disease progression criterion using the EDSS, an EDSS score of less than 6 indicates mild / moderate MS disability, and an EDSS score of 6 or more indicates severe MS disability. As another example, for the MS disability criterion using the PDDS, a PDDS score of 4 or less indicates mild / moderate MS disability, and a PDDS score of more than 4 indicates severe MS disability.

[0056] The terms "marker(s)" and "biomarker(s)" encompass, but are not limited to, lipids, lipoproteins, proteins, cytokines, chemokines, growth factors, peptides, nucleic acids, genes, and oligonucleotides, as well as their associated complexes, metabolites, mutations, variants, polymorphisms, variants, fragments, subunits, degradation products, elements, and other analyte- or sample-derived metrics. Markers can also include mutant proteins, mutant nucleic acids, copy number variations, and / or transcript variants in situations where such mutations, copy number variations, and / or transcript variants are useful in generating predictive models or in predictive models developed using related markers (e.g., non-mutated versions of proteins or nucleic acids, alternative transcripts, etc.).

[0057] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding antibody fragments, e.g., antibodies or antigen-binding fragments thereof, so long as they exhibit the desired biological activity.

[0058] As used herein, an "antibody fragment," and all grammatical variants thereof, is defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, which portion does not contain the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; and any antibody fragment that is a polypeptide having a primary structure consisting of a single contiguous sequence of consecutive amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide").

[0059] The term "biomarker panel" refers to a set of biomarkers that are informative for predicting multiple sclerosis disease activity, and in certain embodiments, for predicting multiple sclerosis disease progression. For example, the expression levels of a set of biomarkers in a biomarker panel can be informative for predicting multiple sclerosis disease progression. In various embodiments, a biomarker panel can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 biomarkers.

[0060] The term "obtaining a dataset associated with a sample" encompasses obtaining a set of data determined from at least one sample. Obtaining a dataset encompasses obtaining a sample and processing the sample to experimentally determine the data. The phrase also encompasses receiving a set of data from a third party, for example, who is processing the sample to experimentally determine the dataset. Additionally, the phrase encompasses mining data from at least one database, or at least one publication, or a combination of a database and a publication. A dataset can be obtained by one of skill in the art through a variety of known means, including being stored in a storage memory.

[0061] It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] II. System Environment Overview 1A shows an overview of a system environment 100 for assessing disease progression in a subject, according to one embodiment. The system environment 100 provides a context for implementing a marker quantification assay 120 and a disease prediction system 130.

[0063] In various embodiments, a test sample is obtained from the subject 110. The sample can be obtained by an individual or a third party, such as a medical professional. Examples of medical professionals include doctors, emergency medical technicians, nurses, first responders, psychologists, phlebotomists, medical physicists, registered nurses, surgeons, dentists, and any other obvious medical professional known to those skilled in the art.

[0064] The test sample is tested to determine the value of one or more markers by performing a marker quantification assay 120. The marker quantification assay 120 determines quantitative expression values ​​of one or more biomarkers in the test sample. The marker quantification assay 120 may be an immunoassay, more specifically, a multiplex immunoassay, examples of which are described in further detail below. Expression levels of various biomarkers can be obtained in a single run using a single test sample obtained from the subject 110. The quantified expression values ​​of the biomarkers are provided to a disease prediction system 130.

[0065] Typically, the disease progression system 130 includes one or more computers implemented as a computer system 700, as described below with respect to Figure 8. Thus, in various embodiments, the steps described with respect to the disease progression system 130 are performed in silico. The disease progression system 130 analyzes biomarker expression values ​​received from the marker quantification assays 120 to generate an assessment 140 of disease progression in the subject 110.

[0066] In various embodiments, the marker quantification assays 120 and the disease progression system 130 may be used by different parties. For example, a first party performs the marker quantification assays 120 and then provides the results to a second party that implements the disease progression system 130. For example, the first party may be a clinical laboratory that obtains a test sample from a subject 110 and performs the assay 120 on the test sample. The second party receives biomarker expression values ​​resulting from the performed assay 120 and analyzes the expression values ​​using the disease progression system 130.

[0067] Reference is now made to Figure 1B, which illustrates a block diagram illustrating the computer logic components of disease progression system 130, according to one embodiment. Specifically, disease progression system 130 may include a model training module 150, a model deployment module 160, and a training data store 170.

[0068] Each of the components of the disease progression system 130 will be described hereinafter in terms of two phases: 1) a training phase and 2) a deployment phase. More specifically, the training phase refers to the construction and training of one or more predictive models based on training data including quantitative expression values ​​of biomarkers obtained from individuals known to be healthy, quiescent, in remission, or in an early stage of disease progression (e.g., mild / moderate MS versus severe MS), or individuals known to be in an active, worsening, relapsing, or more advanced stage of disease progression (e.g., mild / moderate MS versus severe MS). The predictive models are thus trained to predict a subject's disease activity based on the quantitative biomarker expression values. In the deployment phase, the predictive models are applied to quantitative biomarker expression values ​​of test samples obtained from the subjects of interest to generate a prediction of the subject's disease activity.

[0069] In some embodiments, components of disease progression system 130 are applied during one of a training phase and a deployment phase. For example, model training module 150 and training data store 170 (shown by dotted lines in FIG. 1B ) are applied in the training phase, while model deployment module 160 is applied during the deployment phase. In various embodiments, the training and deployment phases may be performed to allow for continuously trained models. For example, model training module 150 may train a model that model deployment module 160 may then deploy. The same model may undergo additional training by model training module 150 (e.g., continuously trained, using new training data as it is acquired). Thus, because the model is continuously trained, it may demonstrate improved predictive ability when analyzing samples during deployment.

[0070] In various embodiments, components of disease progression system 130 may be implemented by different parties depending on whether the components are applied during a training phase or a deployment phase. In such a scenario, the training and deployment of the predictive model are performed by different parties. For example, model training module 150 and training data store 170 applied during the training phase may be used by a first party (e.g., to train the predictive model), and model deployment module 160 applied during the deployment phase may be implemented by a second party (e.g., to deploy the predictive model).

[0071] III. Predictive Model III.A. Training a Predictive Model During the training phase, model training module 150 trains one or more predictive models using training data including expression values ​​of biomarkers. Referring to FIG. 1B , the training data may be stored in training data store 170. In various embodiments, disease progression system 130 generates the training data including expression values ​​of biomarkers by analyzing biomarker expression values ​​in test samples. In various embodiments, disease progression system 130 obtains the training data including expression values ​​of biomarkers from a third party. The third party may have analyzed the test samples to determine the expression values ​​of the biomarkers.

[0072] In various embodiments, training data including biomarker expression values ​​are obtained from clinical subjects. For example, the training data can be biomarker expression values ​​measured from test samples obtained from clinical subjects. Examples of biomarker expression values ​​derived from clinical subjects include biomarker expression values ​​obtained through clinical studies, such as the Multiple Sclerosis CLIMB study (e.g., the Brigham and Women's Hospital Comprehensive Longitudinal Investigation of Multiple Sclerosis), the Accelerated Treatment Project (ACP) for Multiple Sclerosis, and the Expression, Proteomics, Imaging, and Research Clinical (EPIC) study at UCSF and the University Hospital of Basel (UHBC), and the Prospective Investigation of Multiple Sclerosis in the Three Rivers Area (PROMOTE) study at the University of Pittsburgh.

[0073] In various embodiments, the training data further includes reference ground truth indicative of disease activity, such as multiple sclerosis disease activity. By way of example, the training data includes reference ground truth that identifies the presence or absence of multiple sclerosis (MS), relapses or inflammatory events associated with MS, relapse rate (e.g., annual relapse rate), MS status (e.g., worsening or quiescent), response of a subject diagnosed with multiple sclerosis to therapy, degree of multiple sclerosis disability (e.g., a measure of multiple sclerosis disease progression, such as mild, moderate, or severe MS), the risk (e.g., likelihood) of a subject subsequently developing multiple sclerosis, or a measure of minimal disease activity (e.g., the presence or absence of a particular number of gadolinium-enhancing lesions, e.g., the presence or absence of 1, 2, 3, or 4 lesions), or a measure of conventional disease activity (e.g., the presence or absence of one or more gadolinium-enhancing lesions). In certain embodiments, the training data includes a reference ground truth that identifies the degree of multiple sclerosis disability (e.g., a measure of multiple sclerosis disease progression, such as mild, moderate, or severe MS). In various embodiments, the reference ground truth is generated by analyzing images (e.g., brain MRI images, such as T1 or FLAIR images) captured from clinical subjects. Such images may be analyzed via computational means (e.g., image analysis algorithms) or may be analyzed manually. For example, the images may be analyzed to determine brain parenchymal fraction values, which are known markers of MS disease progression. In various embodiments, the brain parenchymal fraction values ​​of the images may serve as the reference ground truth. In various embodiments, the image analysis may be performed by a third party, and the reference ground truth may be used to train the models described herein.

[0074] Reference is made to FIG. 1C , which illustrates an exemplary set of training data 190, according to one embodiment. As shown in FIG. 1C , training data 190 includes data corresponding to multiple individuals (e.g., column 1, which represents individuals 1, 2, 3, 4, ...). For each individual, training data 190 includes quantitative expression values ​​(e.g., A1, B1, A2, B2, etc.) of various biomarkers obtained from the corresponding individual. In some embodiments, the quantitative expression values ​​are determined by marker quantification assay 120 shown in FIG. 1 . While FIG. 1C illustrates four individuals and two different markers (marker A and marker B), training data 190 may include tens, hundreds, or thousands of individuals and tens, hundreds, or thousands of markers.

[0075] As shown in Figure 1C, the first training example of the training data (e.g., row 1) refers to individual 1 and the corresponding quantitative expression value of marker A (e.g., A1) and the corresponding quantitative expression value of marker B (e.g., B1). Similarly, the second training example of the training data (e.g., row 2) refers to individual 2 and the corresponding quantitative expression value of marker A (e.g., A2) and the corresponding quantitative expression value of marker B (e.g., B2). Individuals 3 and 4 have corresponding marker values ​​as shown in Figure 1C.

[0076] As shown in FIG. 1C , training data 190 further includes a reference ground truth (the “Indication” column) that identifies whether the corresponding individual has a positive or negative indication for disease activity. As an example, each indicator may be an indicator of multiple sclerosis disease progression for a patient. For example, referring to the first training example (e.g., row 1), a “positive” indication may reflect the presence of severe disease progression in Individual 1. For example, an MRI scan of Individual 1 may reveal the presence of numerous gadolinium-enhancing lesions. Similarly, a negative result indication (e.g., Individual 3 or Individual 4) reflects the presence of mild or moderate disease progression in the corresponding individual.

[0077] As another example, instead of the reference ground truth indicating a binary option (e.g., positive / negative), the reference ground truth may indicate one of multiple classes. For example, the reference ground truth may include a continuous range of values, each value indicating one of multiple classes. Specifically, the reference ground truth may include a value (e.g., a value of "1") that indicates that the corresponding individual has a presence of mild MS. The reference ground truth may include a value (e.g., a value of "2") that indicates that the corresponding individual has a presence of moderate MS. The reference ground truth may include a value (e.g., a value of "3") that indicates that the corresponding individual has a presence of severe MS. Additional values ​​may be assigned that may further subdivide the disease progression assessment criteria into different classes.

[0078] In various embodiments, the reference ground truth may be a score such as an EDSS score, a PDDS score, a PROMIS score, or an MSRS-R score. Thus, the reference ground truth score itself may be indicative of MS disease progression (e.g., a PDDS score of 4 or less indicates mild / moderate MS, while a PDDS score above 4 indicates severe MS). Thus, by training a predictive model using these reference ground truth scores, the predictive model is trained to predict an individual's score (e.g., an EDSS score, a PDDS score, a PROMIS score, or an MSRS-R score) that is indicative of MS disease progression.

[0079] In various embodiments, the reference ground truth may correspond to brain parenchymal fraction values ​​obtained from images captured from an individual, such as MRI images (T1 or FLAIR images), where brain parenchymal fraction is known to correlate with disease progression in MS patients. Thus, by training a predictive model using these reference ground truth scores, the predictive model is trained to predict values ​​corresponding to brain parenchymal fraction values.

[0080] In various embodiments, the reference ground truth may indicate a particular class according to the brain parenchyma fraction value derived from the MRI image. The MRI images may be analyzed and separated into different subsets according to the brain parenchyma fraction value of the MRI image. For example, the MRI images may be divided into four subsets (e.g., quartiles of brain parenchyma fraction), with the first subset including MRI images with the lowest range of brain parenchyma fraction values, the second subset including MRI images with the next lowest range of brain parenchyma fraction values, the third subset including MRI images with the third lowest range of brain parenchyma fraction values, and the fourth subset including MRI images with the highest range of brain parenchyma fraction values. Thus, by training a predictive model using these reference ground truth scores, the predictive model may be trained to predict different classes depending on the predicted brain parenchyma fraction value.

[0081] In some embodiments, model training module 150 retrieves training data from training data store 170 and randomly divides the training data into a training set and a test set. By way of example, 80% of the training data may be divided into the training set, and the remaining 20% ​​may be divided into the test set. Other ratios of training and test sets may also be implemented. Thus, the training set is used to train the predictive model, while the test set is used to validate the predictive model.

[0082] In various embodiments, the predictive model is any one of a regression model (e.g., linear regression, logistic regression, or polynomial regression), a decision tree, a random forest, a support vector machine, a naive Bayes model, a k-means algorithm, or a neural network (e.g., a feedforward network, a convolutional neural network (CNN), a deep neural network (DNN), an autoencoder neural network, a generative adversarial network, or a recurrent network (e.g., a long short-term memory network (LSTM), a bidirectional recurrent network, a deep bidirectional recurrent network), a linear mixed-effects (LME) model, or any combination thereof. For example, the predictive model can be a stacked classifier that includes both linear regression and a decision tree.

[0083] The predictive model may be trained using any one of machine learning implementations, such as a linear regression algorithm, a logistic regression algorithm, a decision tree algorithm, a support vector machine classification, a naive Bayes classification, a k-nearest neighbor classification, a random forest algorithm, a deep learning algorithm, a gradient boosting algorithm, and a dimensionality reduction technique, such as manifold learning, principal component analysis, factor analysis, autoencoder regularization, and independent component analysis, or a combination thereof. In various embodiments, the cellular disease model is trained using a supervised learning algorithm, an unsupervised learning algorithm, a semi-supervised learning algorithm (e.g., partially supervised), weakly supervised, transfer, multi-task learning, or any combination thereof.

[0084] In various embodiments, a predictive model has one or more parameters, such as hyperparameters or model parameters. Hyperparameters are typically established before training. Examples of hyperparameters include a learning rate, the depth or leaves of a decision tree, the number of hidden layers in a deep neural network, the number of clusters in a k-means cluster, a penalty for a regression model, and a regularization parameter associated with a cost function. Model parameters are typically adjusted during training. Examples of model parameters include weights associated with nodes in a layer of a neural network, support vectors in a support vector machine, and coefficients in a regression model. Model parameters of a cellular disease model are trained (e.g., adjusted) using training data to improve the predictive ability of the cellular disease model.

[0085] Model training module 150 trains one or more predictive models, each of which receives one or more biomarkers as input. In various embodiments, model training module 150 builds a predictive model that receives expression values ​​of two biomarkers as input. In various embodiments, model training module 150 builds a predictive model that receives expression values ​​of three biomarkers as input. In various embodiments, model training module 150 builds a predictive model that receives expression values ​​of four biomarkers as input. In some embodiments, model training module 150 builds a predictive model for more than four biomarkers. For example, a predictive model receives expression values ​​of eight biomarkers as input (e.g., any of the eight biomarkers classified as Tier 1 in Table 2, the eight biomarkers classified as Tier A in Table 1, or the eight biomarkers classified as Tier 1 in Table 3, or their corresponding surrogate biomarkers in Table 4). As another example, the predictive model receives as input the expression values ​​of 17 biomarkers (e.g., the 17 biomarkers classified as Tier 1 or Tier 2 in Table 2, the 17 biomarkers classified as Tier A or Tier B in Table 1, the 17 biomarkers classified as Tier 1 or Tier 2 in Table 3, or any of their corresponding surrogate biomarkers in Table 4). As another example, the predictive model receives as input the expression values ​​of 16 biomarkers (e.g., the 16 biomarkers classified as Tier 1 or Tier 2 in Table 4, excluding COL4A1 in Table 2, or any of their corresponding surrogate biomarkers). As another example, the predictive model receives as input the expression values ​​of 21 biomarkers (e.g., the 21 biomarkers classified as Tier 1, Tier 2, or Tier 3 in Table 2, the 21 biomarkers classified as Tier 1, Tier 2, or Tier 3 in Table 3, the 21 biomarkers classified as Tier A, Tier B, or Tier C in Table 1, or any of their corresponding surrogate biomarkers in Table 4).As another example, the predictive model receives as input the expression values ​​of 20 biomarkers (e.g., any of the 20 biomarkers classified as Tier 1, Tier 2, or Tier 3 in Table 2, excluding COL4A1 in Table 2, or their corresponding surrogate biomarkers in Table 4).

[0086] In various embodiments, the model training module 150 identifies a set of biomarkers to be used to train the predictive model. The model training module 150 may begin with a list of candidate biomarkers that are promising for predicting disease activity (e.g., MS disease progression). In one embodiment, the candidate biomarkers may be biomarkers identified through a literature curation process. In some embodiments, the candidate biomarkers may be biomarkers whose expression values ​​in test samples obtained from individuals positive for disease activity (e.g., the presence of MS, a worsening state, a severe MS state, etc.) are statistically significant compared to the expression values ​​of the biomarkers in test samples obtained from individuals negative for disease activity.

[0087] In one embodiment, model training module 150 performs a feature selection process to identify a set of biomarkers to be included in the biomarker panel. For example, model training module 150 performs sequential forward feature selection based on the expression values ​​of the biomarkers and their importance in predicting a particular endpoint. For example, candidate biomarkers determined to be highly correlated with a particular disease activity endpoint (e.g., a disease progression endpoint) are considered highly important and therefore likely to be included in the biomarker panel compared to other biomarkers that are not highly correlated with the disease activity endpoint (e.g., a disease progression endpoint).

[0088] In some embodiments, the importance of each biomarker for a disease activity endpoint (e.g., a disease progression endpoint) is determined by using one of the following methods: random forest (RF), gradient boosting (GBM), extreme gradient boosting (XGB), or LASSO algorithm. For example, when using the random forest algorithm, the model training module 150 may generate a variable importance plot showing the importance of each candidate biomarker. Specifically, the random forest algorithm may provide, for each candidate biomarker, 1) the average decrease in model accuracy and 2) the average decrease in Gini coefficient, which is a measure of how much each candidate biomarker contributes to the uniformity of the nodes and leaves of the random forest. In one scenario, the importance of each candidate biomarker depends on one or both of the average decrease in model accuracy and the average decrease in Gini coefficient. GBM, XGB, and LASSO may each be used to rank the importance of each candidate biomarker based on its impact value. Thus, the model training module 150 may generate a ranking of each candidate biomarker using one of the following methods: RF, GBM, XGB, or LASSO.

[0089] Each predictive model is iteratively trained using the quantitative expression values ​​of the markers for each individual as input. For example, referring again to FIG. 1C , one iteration involves providing training examples (e.g., rows of training data) including quantitative expression values ​​of biomarkers (e.g., “A1” and “B1”) for a particular individual (e.g., individual 1). Each predictive model is trained with reference ground truth data including indications (e.g., positive or negative results). In various embodiments, over the training iterations, each predictive model is trained (e.g., parameters are adjusted) to minimize the prediction error between the MS activity prediction (e.g., MS disease progression prediction) output by the predictive model and the ground truth data. In various embodiments, the prediction error is calculated based on a loss function, examples of which include an L1 regularized (lasso regression) loss function, an L2 regularized (ridge regression) loss function, or a combination of L1 and L2 regularization (ElasticNet).

[0090] III.B. Deploying Predictive Models During the deployment phase, model deployment module 160 (as shown in FIG. 1B) analyzes quantitative biomarker expression values ​​from test samples obtained from subjects of interest by applying the trained predictive model. In some embodiments, the subjects have not previously been diagnosed with the disease, and thus deployment of the predictive model enables in silico diagnosis of the disease based on quantitative biomarker expression values ​​from the subjects. In some embodiments, the subjects have previously been diagnosed with the disease. As used herein, deployment of the predictive model enables in silico prediction of disease activity (e.g., disease progression) based on quantitative biomarker expression values ​​from the subjects.

[0091] In various embodiments, quantitative biomarker expression values ​​are provided as input to a predictive model. The predictive model analyzes the expression values ​​of the quantitative biomarkers and outputs an assessment of disease activity (e.g., disease progression). The predicted score can provide information about disease activity. For example, the predictive score can allow a subject to be classified into one of multiple disease progression categories (e.g., mild / moderate disease progression or severe progression). In various embodiments, the assessment of disease activity (e.g., disease progression) is a predictive score that represents a learned combination of quantitative biomarker expression values. Generally, the predictive score represents a collection of quantitative expression values ​​and therefore does not directly depend on only one biomarker expression value.

[0092] In various embodiments, the assessment of disease activity is a predictive score that can inform the subject's disease activity. In various embodiments, the predictive score output by the predictive model is compared to one or more reference scores to determine an assessment measure of disease activity. A reference score refers to a previously determined score and is further referred to below as a "healthy score" or "diseased score" corresponding to a diseased or non-diseased patient. For example, one or more scores may be a "healthy score" corresponding to a healthy patient, the patient's own baseline at a previous time point when the patient did not exhibit disease activity (e.g., in a longitudinal analysis), a patient who has been clinically diagnosed with disease but does not exhibit disease activity, or a threshold score (e.g., a cutoff). As another example, one or more scores may be a "diseased score" corresponding to a diseased patient, the patient's own score indicating disease activity at a previous time point, or a threshold score (e.g., a cutoff). As an example, the threshold score may correspond to a healthy patient and may be generated by training a predictive model using the expression values ​​of biomarkers from healthy patients. As another example, the threshold score may correspond to a diseased patient and may be generated by training a predictive model using the expression values ​​of the biomarkers of the diseased patient.

[0093] In various embodiments, the threshold score corresponding to a healthy patient may be lower than the threshold score corresponding to a diseased patient. For example, the threshold score corresponding to a healthy patient may be at least 5% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 10% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 15% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 20% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 25% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 50% lower than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 75% lower than the threshold score corresponding to a diseased patient.

[0094] In various embodiments, the threshold score corresponding to a healthy patient may be higher than the threshold score corresponding to a diseased patient. For example, the threshold score corresponding to a healthy patient may be at least 5% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 10% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 15% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 20% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 25% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 50% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 75% higher than the threshold score corresponding to a diseased patient. As another example, the threshold score corresponding to a healthy patient may be at least 100% higher than the threshold score corresponding to a diseased patient. Thus, in certain embodiments, the prediction score output by the predictive model is compared to one or both of a threshold score corresponding to a healthy patient and a threshold score corresponding to a diseased patient, and an assessment of disease activity is determined based on the comparison.

[0095] In various embodiments, the assessment of disease activity corresponds to the presence or absence of disease. In one embodiment, the predicted score output by the predictive model can be compared to a healthy score. If the subject's predicted score is significantly different from the healthy score (e.g., p-value < 0.05), the subject can be classified as having disease. In one embodiment, the predicted score output by the predictive model can be compared to a diseased score. If the subject's predicted score is significantly different from the diseased score (e.g., p-value < 0.05), the subject can be classified as not having disease. In some embodiments, the predicted score output by the predictive model is compared to both the healthy score and the diseased score. For example, if the subject's predicted score is significantly different from the healthy score (e.g., p-value < 0.05) and not significantly different from the diseased score of a patient diagnosed with disease (e.g., p-value > 0.05), the subject can be classified as having disease. In various embodiments, depending on the subject's classification, the subject can receive treatment. In other words, the assessment can guide the subject's treatment. For example, if a subject is classified as having a disease, the subject may be administered a therapeutic intervention to treat the disease.

[0096] In various embodiments, the assessment of disease activity corresponds to the presence or absence of minimal disease. In one embodiment, the predicted score output by the prediction model can be compared to a score corresponding to an individual previously determined to have minimal disease (e.g., a certain number of gadolinium-enhancing lesions on an MRI scan, e.g., exactly one lesion). If the subject's predicted score is not significantly different (e.g., p-value < 0.05) compared to a score corresponding to an individual previously determined to have minimal disease, the subject can be classified as having minimal disease. If the subject's predicted score is significantly different (e.g., p-value < 0.05) compared to a score corresponding to an individual previously determined to have no minimal disease, the subject can be classified as having minimal disease. In one embodiment, the predicted score output by the prediction model is compared to a score corresponding to an individual without minimal disease (e.g., zero gadolinium-enhancing lesions on an MRI scan). A subject can be classified as not having minimal disease if the subject's predicted score is not significantly different (e.g., p-value > 0.05) from the score corresponding to an individual with no minimal disease (e.g., zero gadolinium-enhancing lesions on the MRI scan). Alternatively, a subject can be classified as having minimal disease if the subject's predicted score is significantly different (e.g., p-value < 0.05) from the score corresponding to an individual with no minimal disease (e.g., zero gadolinium-enhancing lesions on the MRI scan).

[0097] In some embodiments, the predicted score output by the predictive model is compared to both scores corresponding to individuals previously determined to have minimal disease (e.g., a specified number of gadolinium-enhancing lesions on an MRI scan) and scores corresponding to individuals without minimal disease (e.g., zero gadolinium-enhancing lesions on an MRI scan). For example, if the subject's predicted score is significantly different (e.g., p-value < 0.05) compared to the score corresponding to an individual without minimal disease (e.g., zero gadolinium-enhancing lesions on an MRI scan) and is not significantly different (e.g., p-value > 0.05) compared to the score corresponding to an individual previously determined to have minimal disease (e.g., a specified number of gadolinium-enhancing lesions on an MRI scan, e.g., exactly one gadolinium-enhancing lesion), the subject can be classified as having minimal disease.

[0098] In various embodiments, the assessment of disease activity corresponds to the presence or absence of usual disease. In one embodiment, the prediction score output by the prediction model can be compared to a score corresponding to an individual previously determined to have usual disease (e.g., one or more gadolinium-enhancing lesions on an MRI scan). If the subject's prediction score is significantly different (e.g., p-value < 0.05) from a score corresponding to an individual previously determined to have no usual disease, the subject can be classified as having usual disease. If the subject's prediction score is not significantly different (e.g., p-value > 0.05) from a score corresponding to an individual previously determined to have no usual disease, the subject can be classified as not having usual disease. In one embodiment, the prediction score output by the prediction model is compared to a score corresponding to an individual without usual disease (e.g., zero gadolinium-enhancing lesions on an MRI scan). If the subject's prediction score is not significantly different (e.g., p-value > 0.05) from a score corresponding to an individual without usual disease (e.g., zero gadolinium-enhancing lesions on an MRI scan), the subject can be classified as not having usual disease. If the subject's predicted score is significantly different (e.g., p-value < 0.05) from a score corresponding to an individual without usual disease (e.g., zero gadolinium-enhancing lesions on an MRI scan), the subject can be classified as having usual disease. In some embodiments, the predicted score output by the prediction model is compared to both a score corresponding to an individual previously determined to have usual disease (e.g., one or more gadolinium-enhancing lesions on an MRI scan) and a score corresponding to an individual without usual disease (e.g., zero gadolinium-enhancing lesions on an MRI scan). For example, if the subject's predicted score is significantly different (e.g., p-value < 0.05) from a score corresponding to an individual without usual disease (e.g., zero gadolinium-enhancing lesions on an MRI scan) and is not significantly different (e.g., p-value > 0.05) from a score corresponding to an individual previously determined to have usual disease (e.g., one or more gadolinium-enhancing lesions on an MRI scan), the subject can be classified as having usual disease.

[0099] In various embodiments, the assessment of disease activity corresponds to a directional shift in disease activity based on a predicted increase or decrease in the number of gadolinium-enhancing lesions. In one embodiment, the prediction score output by the prediction model can be compared to a score corresponding to an individual previously determined to be experiencing increased disease activity (e.g., an increase in the number of gadolinium-enhancing lesions on an MRI scan). If the subject's prediction score is significantly different (e.g., p-value < 0.05) from a score corresponding to an individual previously determined not to be experiencing increased disease activity, the subject can be classified as likely to experience increased disease activity. If the subject's prediction score is not significantly different (e.g., p-value > 0.05) from a score corresponding to an individual previously determined not to be experiencing increased disease activity, the subject can be classified as unlikely to experience increased disease activity. In one embodiment, the prediction score output by the prediction model is compared to a score corresponding to an individual previously determined to be experiencing decreased disease activity (e.g., a gradual decrease in the number of gadolinium-enhancing lesions on an MRI scan). If the subject's predicted score is not significantly different (e.g., p-value > 0.05) from scores corresponding to individuals experiencing a decrease in disease activity, the subject can be classified as likely to experience a decrease in disease activity. If the subject's predicted score is significantly different (e.g., p-value > 0.05) from scores corresponding to individuals experiencing a decrease in disease activity, the subject can be classified as likely to experience a decrease in disease activity. In some embodiments, the predicted score output by the predictive model is compared to both scores corresponding to individuals previously determined to be experiencing increased disease activity (e.g., a gradual increase in the number of gadolinium-enhancing lesions on MRI scans) and scores corresponding to individuals experiencing a decrease in disease activity (e.g., a gradual decrease in the number of gadolinium-enhancing lesions on MRI scans).For example, a subject can be classified as likely to experience increased disease activity if their predicted score is significantly different (e.g., p-value < 0.05) compared to scores corresponding to individuals experiencing a decrease in disease activity (e.g., a gradual decrease in the number of gadolinium-enhancing lesions on an MRI scan) and not significantly different (e.g., p-value > 0.05) compared to scores corresponding to individuals experiencing an increase in disease activity (e.g., a gradual increase in the number of gadolinium-enhancing lesions on an MRI scan). In various embodiments, a subject can be classified as unlikely to experience either an increase or decrease in disease activity (e.g., the subject's disease activity is stable) if their predicted score is not significantly different (e.g., p-value > 0.05) compared to scores corresponding to both individuals experiencing an increase in disease activity and individuals experiencing a decrease in disease activity.

[0100] In various embodiments, the assessment of disease activity corresponds to the state of the disease in the subject. For example, if the disease is MS, the state of the disease in the subject is quiescent versus worsening. In one embodiment, the predicted score output by the prediction model can be compared to scores corresponding to individuals previously determined to be in a quiescent state (e.g., previously determined to be clinically quiescent). If the subject's predicted score is significantly different (e.g., p-value < 0.05) from scores corresponding to individuals previously determined not to be in a quiescent state, the subject can be classified as being in a quiescent state. If the subject's predicted score is not significantly different (e.g., p-value > 0.05) from scores corresponding to individuals previously determined to be in a quiescent state, the subject can be classified as not being in a quiescent state. In one embodiment, the predicted score output by the prediction model is compared to scores corresponding to individuals previously determined to be worsening. If the subject's predicted score is not significantly different (e.g., p-value > 0.05) from scores corresponding to individuals previously determined to be worsening, the subject can be classified as being worsening. If the subject's predicted score is significantly different (e.g., p-value < 0.05) from the score corresponding to an individual previously determined to be in a deteriorated state, the subject can be classified as not in a deteriorated state. In some embodiments, the predicted score output by the predictive model is compared to both the score corresponding to an individual previously determined to be in a quiescent state and the score corresponding to an individual in a deteriorated state. For example, if the subject's predicted score is significantly different (e.g., p-value < 0.05) from the score corresponding to an individual previously determined to be in a deteriorated state, the subject can be classified as being in a deteriorated state.

[0101] In various embodiments, the assessment of disease activity corresponds to a likely response to a therapy provided to the subject. In one embodiment, the predicted score output by the predictive model can be compared to a score corresponding to an individual previously determined to be responsive to the therapy (e.g., previously clinically determined to be responsive to the therapy). If the subject's predicted score is significantly different (e.g., p-value < 0.05) compared to a score corresponding to an individual previously determined not to be responsive to the therapy, the subject can be classified as a responder. If the subject's predicted score is not significantly different (e.g., p-value > 0.05) compared to a score corresponding to an individual previously determined to be responsive to the therapy, the subject can be classified as a responder. In one embodiment, the predicted score output by the predictive model is compared to a score corresponding to an individual previously determined to be a non-responder. If the subject's predicted score is not significantly different (e.g., p-value > 0.05) from a score corresponding to an individual previously determined to be a non-responder, the subject can be classified as a non-responder. If the subject's predicted score is significantly different (e.g., p-value < 0.05) from scores corresponding to individuals previously determined to be responders, the subject can be classified as a non-responder. In some embodiments, the predicted score output by the predictive model is compared to both scores corresponding to individuals previously determined to be responders and scores corresponding to individuals previously determined to be non-responders. For example, if the subject's predicted score is significantly different (e.g., p-value < 0.05) from scores corresponding to individuals previously determined to be responders, and is significantly different (e.g., p-value > 0.05) from scores corresponding to individuals previously determined to be responders, the subject can be classified as a responder.

[0102] In various embodiments, the assessment of disease activity is a classification of disease progression (e.g., mild / moderate disease vs. severe disability). Thus, in such embodiments, the predicted score output by the predictive model can be compared to one or both of scores corresponding to individuals previously identified as having mild / moderate disease and scores corresponding to individuals previously identified as having severe disability.

[0103] In various embodiments, a score corresponding to an individual previously identified as having a mild / moderate disease may be lower than a score corresponding to an individual previously identified as having a severe disorder. For example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 5% lower than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 10% lower than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 15% lower than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 20% lower than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 25% lower than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having mild / moderate disease may be at least 50% lower than a score corresponding to an individual previously identified as having severe disability. As another example, a score corresponding to an individual previously identified as having mild / moderate disease may be at least 75% lower than a score corresponding to an individual previously identified as having severe disability.

[0104] In various embodiments, a score corresponding to an individual previously identified as having a mild / moderate disease may be higher than a score corresponding to an individual previously identified as having a severe disorder. For example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 5% higher than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 10% higher than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 15% higher than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 20% higher than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having a mild / moderate disease may be at least 25% higher than a score corresponding to an individual previously identified as having a severe disorder. As another example, a score corresponding to an individual previously identified as having mild / moderate disease may be at least 50% higher than a score corresponding to an individual previously identified as having severe disability. As another example, a score corresponding to an individual previously identified as having mild / moderate disease may be at least 75% higher than a score corresponding to an individual previously identified as having severe disability. As another example, a score corresponding to an individual previously identified as having mild / moderate disease may be at least 100% higher than a score corresponding to an individual previously identified as having severe disability. Thus, in certain embodiments, the predicted score output by the predictive model is compared to one or both of the scores corresponding to individuals previously identified as having mild / moderate disease and the scores corresponding to individuals previously identified as having severe disability, and an assessment of disease progression is determined based on the comparison.

[0105] In one embodiment, the assessment of disease activity is an assessment of disease progression and may correspond to the degree of MS disability in a subject diagnosed with multiple sclerosis. In one embodiment, the degree of MS disability corresponds to an EDSS score or a range of EDSS scores. In various embodiments, the assessment (e.g., predicted score) corresponding to a subject is compared to multiple reference scores. Each reference score may correspond to a group of individuals clinically classified by degree of disability. In various embodiments, the reference score is an EDSS score. In various embodiments, the reference score corresponds to an EDSS score. For example, a first reference score may correspond to an individual clinically classified with an EDSS score of 1. Additional reference scores may correspond to groups of individuals clinically classified with scores of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0. As another example, a first reference score may correspond to individuals previously classified as having a score between 1 and 6 on the EDSS scale, and a second reference score may correspond to individuals previously classified as having a score between 6.5 and 10 on the EDSS scale. In one scenario, if a subject's predicted score output by a predictive model is not significantly different from one group (e.g., p-value > 0.05) and is significantly different compared to all other groups (e.g., p-value < 0.05), the subject may be classified into one of the EDSS scores. The subject may be treated according to a clinical protocol based on the classification.

[0106] In some embodiments, the degree of MS disability corresponds to a PDDS score or a range of PDDS scores. In various embodiments, the assessment (e.g., predicted score) corresponding to a subject is compared to multiple scores. Each score can correspond to a group of individuals clinically classified by degree of disability. In various embodiments, the reference score is a PDDS score. In various embodiments, the reference score corresponds to a PDDS score. For example, a first reference score can correspond to individuals clinically classified with a score of 1 on the PDDS scale. Additional reference scores can correspond to groups of individuals previously classified with a score of 2, 3, 4, 5, 6, 7, or 8. As another example, a first reference score can correspond to individuals previously classified with a score between 1 and 4 on the PDDS scale, and a second reference score can correspond to individuals previously classified with a score between 5 and 8 on the PDDS scale. In one scenario, if the predicted score of a subject output by the predictive model is not significantly different from one group (e.g., p-value > 0.05) and is significantly different from the other group (e.g., p-value < 0.05), the subject can be classified into one of the PDDS scores. The subject can be treated according to a clinical protocol based on the classification.

[0107] In some embodiments, the degree of MS disability corresponds to the brain parenchymal fraction score. In various embodiments, the assessment (e.g., predicted score) corresponding to the subject is compared to one or more reference scores. For example, a first reference score can be a brain parenchymal fraction score corresponding to an individual previously classified as having mild / moderate MS. A second reference score can be a brain parenchymal fraction score corresponding to an individual previously classified as having severe MS. In one scenario, if the subject's predicted score output by the predictive model is not significantly different from one group (e.g., p-value > 0.05) and is significantly different from all other groups (e.g., p-value < 0.05), the subject can be classified as having mild / moderate or severe MS. The subject can be treated according to a clinical protocol based on the classification.

[0108] In some embodiments, the degree of MS disability corresponds to a PROMIS score. In various embodiments, the assessment (e.g., predicted score) corresponding to a subject is compared to one or more reference scores. For example, a first reference score can be a PROMIS score corresponding to an individual previously classified as having mild / moderate MS. A second reference score can be a PROMIS score corresponding to an individual previously classified as having severe MS. In one scenario, if the subject's predicted score output by the predictive model is not significantly different from one group (e.g., p-value > 0.05) and is significantly different from all other groups (e.g., p-value < 0.05), the subject can be classified as having mild / moderate or severe MS. The subject can be treated according to a clinical protocol based on the classification.

[0109] In some embodiments, the degree of MS disability corresponds to the MSRS-R score. In various embodiments, the assessment (e.g., predicted score) corresponding to the subject is compared to one or more reference scores. For example, a first reference score can be an MSRS-R score corresponding to an individual previously classified as having mild / moderate MS. A second reference score can be an MSRS-R score corresponding to an individual previously classified as having severe MS. In one scenario, if the subject's predicted score output by the predictive model is not significantly different from one group (e.g., p-value > 0.05) and is significantly different from all other groups (e.g., p-value < 0.05), the subject can be classified as having mild / moderate or severe MS. The subject can be treated according to a clinical protocol based on the classification.

[0110] In one embodiment, the assessment of disease activity corresponds to the subject's risk (e.g., likelihood) of subsequently developing the disease. In various embodiments, the assessment (e.g., prediction score) corresponding to the subject is compared to multiple scores. Each score may correspond to a group of individuals within a risk group that has been clinically classified at a particular risk of developing MS. As an example, risk groups may be divided into high-risk, intermediate-risk, and low-risk groups. In one scenario, a subject may be classified into a risk group if their prediction score is not significantly different from one group (e.g., p-value > 0.05) and is significantly different from the other groups (e.g., p-value < 0.05). Thus, the subject can make lifestyle and / or treatment changes based on the predicted risk / likelihood of developing MS.

[0111] In various embodiments, the disease activity measures predicted by the predictive model provide additional utility for managing a patient's disease activity. By way of example, the disease activity measures predicted by the predictive model are useful for selecting candidate therapeutics or for determining the effectiveness of previously administered therapeutics.

[0112] In various embodiments, the disease activity measure predicted by the predictive model for a patient can be compared to a prior measure of disease activity to determine whether a therapeutic agent administered to the patient is effective. As one example, the prior measure of disease activity may be a prediction determined for the same patient (e.g., a baseline measure of disease activity). Thus, in this example, the comparison of the disease activity measure and the prior measure of disease activity is a longitudinal analysis of patients undergoing treatment with a therapy. As such, a difference, or lack of difference, between the disease activity measure and the prior measure of disease activity may indicate that the therapeutic agent is having an effect or is ineffective. As another example, the prior measure of disease activity may be a measure determined for a patient population (e.g., a reference set of patients). In this example, the comparison of the disease activity measure and the prior measure of disease activity may reveal whether the patient is experiencing a benefit from the therapeutic agent (evidenced by the disease activity measure) compared to the prior measure of disease activity for the patient population.

[0113] In various embodiments, if a comparison between the disease activity assessment and a previous assessment of disease activity indicates that a currently administered therapeutic agent is ineffective or not as effective as desired, a change in the patient's treatment can be made. In one embodiment, the treatment administration of a currently administered therapeutic agent can be changed to effect a patient response. For example, a currently administered therapeutic agent can be increased in dosage. In one embodiment, a candidate therapeutic agent can be selected for administration to the patient. In various embodiments, the candidate therapeutic agent can be administered to the patient in place of a currently administered therapeutic agent, or the candidate therapeutic agent can be administered to the patient in addition to a currently administered therapeutic agent.

[0114] As another example, disease activity metrics are useful in supporting symptom and medication tracking, nursing interventions, laboratory monitoring, and controlled longitudinal MRI reporting. In such scenarios, disease activity metrics may reduce unplanned healthcare utilization (e.g., unplanned clinic visits), thereby improving patient and physician satisfaction.

[0115] IV. Biomarker Panel In various embodiments, assessing disease activity (e.g., disease progression) involves implementing a univariate biomarker panel. Thus, the univariate biomarker panel includes one biomarker. In other embodiments, assessing disease activity (e.g., disease progression) involves implementing a multivariate biomarker panel. In such embodiments, the multivariate biomarker panel includes multiple biomarkers. In various embodiments, the multivariate biomarker panel includes two biomarkers. In various embodiments, the multivariate biomarker panel comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 biomarkers. In certain embodiments, the multivariate biomarker panel comprises two biomarkers. In certain embodiments, the multivariate biomarker panel comprises three biomarkers. In certain embodiments, the multivariate biomarker panel comprises four biomarkers. In certain embodiments, the multivariate biomarker panel comprises seven biomarkers. In certain embodiments, the multivariate biomarker panel comprises eight biomarkers. In certain embodiments, the multivariate biomarker panel comprises 16 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 17 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 20 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 21 biomarkers.

[0116] In certain embodiments described herein, biomarker panels are implemented for the assessment or prediction of disease progression, such as MS disease progression. In various embodiments, assessing disease progression involves implementing a univariate biomarker panel. Thus, the univariate biomarker panel includes one biomarker. In other embodiments, assessing disease progression involves implementing a multivariate biomarker panel. In such embodiments, the multivariate biomarker panel for assessing disease progression includes multiple biomarkers. In various embodiments, the multivariate biomarker panel for assessing disease progression includes two biomarkers. In various embodiments, a multivariate biomarker panel for assessing disease progression comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 biomarkers. In certain embodiments, the multivariate biomarker panel comprises two biomarkers. In certain embodiments, the multivariate biomarker panel comprises three biomarkers. In certain embodiments, the multivariate biomarker panel comprises four biomarkers. In certain embodiments, the multivariate biomarker panel comprises seven biomarkers. In certain embodiments, the multivariate biomarker panel comprises 8 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 16 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 17 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 20 biomarkers. In certain embodiments, the multivariate biomarker panel comprises 21 biomarkers.

[0117] In various embodiments, the multivariate biomarker panel further incorporates one or more subject attributes, which may include, for example, subject age, subject sex, disease duration experienced by the subject (e.g., disease duration of MS), racial / ethnic identity, weight, height, body mass index (BMI), and socioeconomic status.

[0118] In one embodiment, the biomarkers in the biomarker panel may include one or more of the following: 6Ckine, adiponectin, adrenomedullin (ADM), alpha 1 antitrypsin (AAT), alpha 1 microglobulin (A1Micro), alpha 2 macroglobulin (A2Macro), alpha fetoprotein (AFP), amphiregulin (AR), angiogenin, angiopoietin 1 (ANG-1), angiopoietin 2 (ANG-2), angiotensin converting enzyme (ACE), anti-leukoproteinase (ALP), antithrombin III (AT). III), apolipoprotein A (Apo-A), apolipoprotein D (Apo-D), apolipoprotein E (Apo-E), AXL receptor tyrosine kinase (AXL), B cell activating factor (BAFF), B lymphocyte chemoattractant (BLC), beta amyloid (1-40) (AB-40), beta amyloid (1-42) (AB-42), beta 2 microglobulin (B2M), beta cellulin (BTC), brain-derived neurotrophic factor (BDNF), C-reactive protein (CRP), cadherin 1 (E-Cad), calbindin, cancer antigen 125 (CA-125), cancer antigen 15-3 (CA15-3), cancer antigen 19-9 (CA19-9), carbonic anhydrase 9 (CA-9), carcinoembryonic antigen (CEA), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), cathepsin D, CD40 ligand (CD40-L), CD163, ceruloplasmin, chemokine CC-4 (HCC-4), chromogranin A (CgA), ciliary neurotrophic factor (CNTF), clusterin (CLU), complement C3 (C3), complement factor H (CFH), complement factor H-related protein 1 (CFHR1), cystatin B, cystatin C, decorin, Dickkop f-related protein 1 (DKK-1), dopamine β-hydroxylase (DBH), E-selectin, EN-RAGE, eotaxin 1, eotaxin 2, eotaxin 3, epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), epiregulin (EPR), epithelial-derived neutrophil-activating protein 78 (ENA-78), erythropoietin (EPO), factor VII, Fas ligand (FasL), FASLG receptor (FAS), ferritin (FRTN), fibrinogen, fibrin 1C (Fib1C), ficolin 3, follicle-stimulating hormone (FSH),Gastric inhibitory polypeptide (GIP), gelsolin, glucagon-like peptide-1 (GLP-1), glycogen phosphorylase isoenzyme BB (GPBB), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor 15 (GDF-15), growth hormone (GH), growth regulatory alpha protein (GRO-α), haptoglobin, heat shock protein 70 (HSP-70), heparin-binding EGF-like growth factor (HB-EGF), hepatocyte growth factor (HGF), human chorionic gonadotropin beta (hCG) CG), immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin M (IgM), insulin, insulin-like growth factor binding protein 2 (IGFBP2), intercellular adhesion molecule 1 (ICAM-1), interferon α (IFN-α), interferon γ (IFN-γ), interferon γ-inducible protein 10 (IP-10), interferon-inducible T cell α chemoattractant (ITAC), interleukin 1 α (IL-1α), interleukin 1 β (IL-1β), interleukin 1 receptor antagonist (IL1 ra), interleukin 2 (IL-2), interleukin 2 receptor α (IL2 receptor α), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 6 receptor (IL6r), interleukin 6 receptor subunit β (IL6Rβ), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 subunit p40 (IL12p40), interleukin 12 subunit p70 (IL12p70), interleukin 13 (IL13), interleukin 15 (IL15), interleukin 16 (IL16), interleukin 17 (IL17), interleukin 18 (IL18), interleukin 18 binding protein (IL18bp), interleukin 22 (IL22), interleukin 23 (IL23), interleukin 31 (IL31), kidney injury molecule 1 (KIM-1), lactoferrin (LTF), latency-associated peptide of transforming growth factor-β1 (LAP TGF b1), leptin, leptin receptor (leptin R),Leucine-rich α2-glycoprotein (LRG1), luteinizing hormone (LH), macrophage colony-stimulating factor 1 (M-CSF), macrophage-derived chemokine (MDC), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β), macrophage inflammatory protein 3α (MIP-3α), macrophage inflammatory protein 3β (MIP-3β), macrophage migration inhibitory factor (MIF), macrophage-stimulating protein (MSP), mast stem cell growth factor receptor (SCFR), matrix metalloproteinase (MTC) Metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 3 (MMP-3), matrix metalloproteinase 7 (MMP-7), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 9 total (MMP-9 total), matrix metalloproteinase 10 (MMP-10), microalbumin, monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein Mitotic protein 4 (MCP-4), monokine-induced by gamma interferon (MIG), myeloid progenitor inhibitory factor 1 (MPIF-1), myeloperoxidase (MPO), myoglobin, nerve growth factor beta (NGF-β), neurofilament heavy polypeptide (NF-H), neuron-specific enolase (NSE), neuronal cell adhesion molecule (NrCAM), neuropilin-1, neutrophil-activating peptide 2 (NAP-2), omentin, osteocalcin, osteopontin, osteoprotegerin (OPG), P-selectin, pancreatic polypeptide PPP, pancreatic secretory trypsin inhibitor (TATI), paraoxonase-1 (PON1), pepsinogen-I (PGI), periostin, pigment epithelium-derived factor (PEDF), placental growth factor (PLGF), plasminogen activator inhibitor 1 (PAI-1), platelet endothelial cell adhesion molecule (PECAM-1), platelet-derived growth factor BB (PDGF-BB), prolactin (PRL), prostate-specific antigen-free (PSA-f), protein DJ-1 (DJ-1), lung and activation-regulated chemokine (PARC), pulmonary surfactant protein D (SP-D),Receptor for advanced glycation end products (RAGE), resistin, S100 calcium-binding protein B (S100B), serum amyloid A protein (SAA), serum amyloid P component (SAP), sex hormone-binding globulin (SHBG), sortilin, ST2, stem cell factor (SCF), stromal cell-derived factor 1 (SDF-1), superoxide dismutase 1 soluble (SOD-1), T cell-specific protein RANTES (RANTES), T lymphocyte-secreted protein I309 (I309), Tamm-Horsfall glycoprotein (THP), tenascin-C (TN-C), tetranectin, thrombin-activated fibronectin (TAFI), thrombospondin 1, thymus- and activation-regulated chemokine (TARC), thyroid-stimulating hormone (TSH), thyroxine-binding globulin (TBG), tissue inhibitor of metalloproteinases 1 (TIM) P-1), tissue inhibitor of metalloproteinases 2 (TIMP-2), TNF-related apoptosis-inducing ligand receptor 3 (TRAIL-R3), transferrin receptor protein 1 (TFR1), transforming growth factor beta 3 (TGF-β3), tumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β), tumor necrosis factor ligand superfamily member 12 (Tweak), tumor necrosis factor ligand superfamily member 13 (APRIL), tumor necrosis factor receptor I (TNF-RI), tumor necrosis factor receptor 2 (TNFR2), vascular cell adhesion molecule 1 (VCAM-1), vascular endothelial growth factor (VEGF), visceral adipose tissue-derived serpin A12 (Vaspin), visfatin, vitamin D binding protein (VDBP), vitronectin, von Willebrand factor (vWF), or YKL-40.

[0119] In some embodiments, the biomarkers of the biomarker panel include the biomarkers set forth in Tables 4-6. In some embodiments, the biomarkers may include one or more of the following: neurofilament light polypeptide chain (NEFL), myelin oligodendrocyte glycoprotein (MOG), cluster of differentiation 6 (CD6), chemokine (C-X-C motif) ligand 9 (CXCL9), osteoprotegerin (OPG), osteopontin (OPN), matrix metallopeptidase 9 (MMP-9), glial fibrillary acidic protein (GFAP), C-UB domain-containing protein 1 (CDCP1), C-C motif chemokine ligand 20 (CCL20 / MIP-3α). , interleukin-12 subunit beta (IL-12B), amyloid beta precursor-like protein 1 (APLP1), tumor necrosis factor receptor superfamily member 10A (TNFRSF10A), collagen, type IV, alpha 1 (COL4A1), serpin family A member 9 (SERPINA9), fibronectin leucine-rich transmembrane protein 2 (FLRT2), chemokine (C-X-C motif) ligand 13 (CXCL13), growth hormone (GH), versican core protein (VCAN), protogenin (PRTG), contactin 2 (CNTN2). In some embodiments, the biomarkers further include growth hormone (GH2), interleukin-18 (IL18), matrix metalloproteinase 2 (MMP-2), gamma-interferon-inducible lysosomal thiol reductase (IFI30), and chitinase 3-like protein 1 (CHI3L1 / YkL40).

[0120] In some embodiments, the biomarkers may include one or more of the following: cell adhesion molecule 3 (CADM3), kallikrein-related peptidase 6 (KLK6), brevican (BCAN), oligodendrocyte myelin glycoprotein (OMG), CD5 molecule (CD5), cytotoxic and regulatory T cell (CRTAM), CD244 molecule (CD244), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), proteinase 3 (PRTN3), follistatin-like 3 (FSTL3), C-X-C motif chemokine ligand 10 (CXCL1), and / or CXC motif chemokine ligand 10 (CXCL1). 0), C-X-C motif chemokine ligand 11 (CXCL11), interleukin-18 binding protein (IL-18BP), macrophage scavenger receptor 1 (MSR1), C-C motif chemokine ligand 3 (CCL3), tumor necrosis factor ligand superfamily member 12 (TWEAK), trefoil factor 3 (TFF3), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2), insulin-like growth factor binding protein 1 (IGFBP-1), interleukin-12A (IL12A), and stroke-related 6 homolog-like (SEZ6L), dipeptidyl peptidase-like 6 (DPP6), neurocan (NCAN), tubulointerstitial nephritis antigen-like 1 (TINAGL1), calcium-activated nucleotidase 1 (CANT1), nectin cell adhesion molecule 2 (NECTIN2), neural proliferation, differentiation, and regulatory protein 1 (NPDC1), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A), contactin 4 (CNTN4), neurotrophic receptor tyrosine kinase 2 (NTRK2), neurotrophic receptor tyrosine kinase 3 (NTRK3), cadherin 6 (CD H6), carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8), mitotic arrest deficient 1-like 1 (MAD1L1), Fc fragment of IgA receptor (FCAR), myeloperoxidase (MPO), osteomodulin (OMD), matrix extracellular phosphoglycoprotein (MEPE), GDNF family receptor α3 (GDNFR-α3), scavenger receptor class F member 2 (SCARF2), CD40 ligand (IgM), tumor necrosis factor receptor superfamily member 1B (TNF-R2), programmed cell death 1 ligand (PD-L1),Notch3 (NOTCH3), contactin 1 (CNTN1), oncostatin M (OSM), transforming growth factor α (TGF-α), peptidoglycan recognition protein 1 (PGLYRP1), nitric oxide synthase 3 (NOS3).

[0121] In certain embodiments, a biomarker panel useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) includes biomarkers identified as any of Tier A of Table 1, Tier 1 of Table 2, Tier 1 of Table 3, or their corresponding surrogate biomarkers in Table 4. For example, the biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, MMP-9, and GFAP (Tier A of Table 1). As another example, the biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, and GFAP (Tier 1 of Table 2). As another example, the biomarker panel includes GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, and NEFL (Tier 1 of Table 3).

[0122] In certain embodiments, a biomarker panel useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) includes biomarkers identified as any of Tier B of Table 1, Tier 2 of Table 2, Tier 2 of Table 3, or the corresponding surrogate biomarkers of Table 4. For example, the biomarker panel includes CDCP1, CCL20 / MIP 3-α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, and CXCL13 (Tier B of Table 1). As another example, the biomarker panel includes CDCP1, CCL20 / MIP-3α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, and TNFSF13B (Tier 2 of Table 2). As another example, the biomarker panel includes CDCP1, CCL20 / MIP3-α, IL-12B, APLP1, TNFRSF10A, SERPINA9, FLRT2, and TNFSF13B (all biomarkers listed as Tier 2 in Table 2 except for COL4A1). As another example, the biomarker panel includes CXCL9, TNFRSF10A, CCL20 / MIP-3α, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, and CNTN2 (all biomarkers listed as Tier 2 in Table 3).

[0123] In certain embodiments, a biomarker panel useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) includes biomarkers identified as either Tier C of Table 1, Tier 3 of Table 2, Tier 3 of Table 3, or their corresponding surrogate biomarkers in Table 4. For example, the biomarker panel includes GH, VCAN, PRTG, and CNTN2 (Tier C of Table 1 and Tier 3 of Table 2). For example, the biomarker panel includes COL4A1, GH, IL-12B, and PRTG (Tier 3 of Table 3).

[0124] In certain embodiments, a biomarker panel useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) includes biomarkers identified as any of Tier A and Tier B in Table 1, Tier 2 in Table 2, Tier 1 and Tier 2 in Table 3, or the corresponding surrogate biomarkers in Table 4. For example, the biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, MMP-9, GFAP, CDCP1, CCL20 / MIP 3-α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, and CXCL13 (Tier A and B in Table 1). As another example, a biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20 / MIP-3α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, and TNFSF13B (Tier 1 and 2 in Table 2). As another example, a biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20 / MIP-3α, IL-12B, APLP1, TNFRSF10A, SERPINA9, FLRT2, and TNFSF13B (all biomarkers listed as Tier 1 and 2 in Table 2 except for COL4A1). As another example, a biomarker panel may include GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-α, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, and CNTN2 (all biomarkers listed as Tier 1 and 2 in Table 3).

[0125] In certain embodiments, a biomarker panel useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) includes biomarkers identified as any of Tier A, Tier B, or Tier C of Table 1, Tier 1, Tier 2, or Tier 3 of Table 2, Tier 1, Tier 2, and Tier 3 of Table 3, or their corresponding surrogate biomarkers in Table 4. For example, the biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, MMP-9, GFAP, CDCP1, CCL20 / MIP 3-α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, CXCL13, GH, VCAN, PRTG, and CNTN2 (Tiers A, B, and C of Table 1). As another example, a biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20 / MIP-3α, IL-12B, APLP1, TNFRSF10A, COL4A1, SERPINA9, FLRT2, TNFSF13B, GH, VCAN, PRTG, CNTN2, GH2, IL18, MMP-2, IFI30, and CHI3L1 / YkL40 (Tiers 1, 2, and 3 of Table 2). As another example, a biomarker panel includes NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20 / MIP-3α, IL-12B, APLP1, TNFRSF10A, SERPINA9, FLRT2, TNFSF13B, GH, VCAN, PRTG, CNTN2, GH2, IL18, MMP-2, IFI30, and CHI3L1 / YkL40 (all biomarkers listed in Tiers 1, 2, and 3 of Table 2 except for COL4A1). As another example, a biomarker panel includes GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-α, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG (Tiers 1, 2, and 3 of Table 3).

[0126] In various embodiments, a biomarker panel for generating a prediction (e.g., a prediction of disease activity or a prediction of disease progression) includes a minimum set of predictive biomarkers, e.g., a biomarker pair, a biomarker triad, or a biomarker quad. In various embodiments, at least one of the biomarkers in the biomarker pair, biomarker triad, or biomarker quad is NEFL. In various embodiments, at least one of the biomarkers in the biomarker pair, biomarker triad, or biomarker quad is MOG. In various embodiments, the biomarker pair, biomarker triad, or biomarker quad does not include NEFL. In such embodiments, the biomarker pair, biomarker triad, or biomarker quad that does not include NEFL includes MOG.

[0127] Examples of biomarker pairs useful for generating predictions (e.g., predictions of MS disease activity or predictions of MS disease progression) include 1) NEFL and MOG, 2) NEFL and CD6, 3) NEFL and CXCL9, 4) NEFL and TNFSF10A, 5) MOG and IL-12B, 6) CXCL9 and CD6, 7) MOG and CXCL9, 8) MOG and CD6, 9) CXCL9 and COL4A1, and 10) CD6 and VCAN. Additional examples of biomarker pairs predicting multiple sclerosis disease activity include 1) NEFL and TNFSF13B, 2) NEFL and CNTN2, 3) NEFL and CXCL9, 4) MOG and CDCP1, 5) MOG and TNFSF13B, and 6) MOG and CXCL9. Additional examples of biomarker pairs that predict multiple sclerosis disease activity include 1) NEFL and TNFSF13B, 2) NEFL and SERPINA9, 3) NEFL and GH, 4) MOG and TNFSF13B, 5) MOG and CXCL9, and 6) MOG and IL-12B.

[0128] Examples of biomarker triads useful for generating a prediction (e.g., a prediction of MS disease activity or a prediction of MS disease progression) include: 1) MOG, IL-12B, and APLP1; 2) MOG, CD6, and CXCL9; 3) CXCL9, COL4A1, and VCAN; 4) NEFL, CD6, and CXCL9; 5) NEFL, TNFRSF10A, and COL4A1; 6) MOG, IL-12B, and CNTN2; and 7) CD6, CCL20, and VCAN. Additional examples of biomarker triads that predict multiple sclerosis disease activity include: 1) NEFL, CNTN2, and TNFSF13B; 2) NEFL, APLP1, and TNFSF13B; 3) NEFL, TNFRSF10A, and TNFSF13B; 4) MOG, CXCL9, and TNFSF13B; 5) MOG, OPG, and TNFSF13B; and 6) MOG, CCL20, and TNFSF13B. Additional examples of biomarker triads that predict multiple sclerosis disease activity include: 1) NEFL, SERPINA9, and TNFSF13B; 2) NEFL, CNTN2, and TNFSF13B; 3) NEFL, APLP1, and TNFSF13B; 4) MOB, CXCL9, and TNFSF13B; 5) MOG, SERPINA9, and TNFSF13B; and 6) MOG, OPG, and TNFSF13B.

[0129] Examples of biomarker tetrads useful for generating predictions (e.g., predictions of MS disease activity or predictions of MS disease progression) include: 1) NEFL, MOG, CD6, and CXCL9; 2) NEFL, CXCL9, TNFRSF10A, and COL4A1; 3) MOG, CXCL9, IL-12B, and APLP1; 4) CXCL9, COL4A1, OPG, and VCAN; 5) CXCL9, OPG, APLP1, and OPN; 6) NEFL, CD6, CXCL9, and CXCL13; 7) NEFL, MOG, CD6, and CXCL9; 8) NEFL, TNFRSF10A, COL4A1, and CCL20; 9) MOG, IL-12B, OPN, and CNTN2; and 10) CD6, COL4A1, CCL20, and VCA. Additional examples of biomarker tetrads that predict multiple sclerosis disease activity include: 1) NEFL, TNFRSF10A, CNTN2, and TNFSF13B; 2) NEFL, COL4A1, CNTN2, and TNFSF13B; 3) NEFL, TNFRSF10A, APLP1, and TNFSF13B; 4) MOG, CXCL9, APLP1, and TNFSF13B; 5) MOG, CXCL9, OPG, and TNFSF13B; and 6) MOG, CXCL9, OPG, and CNTN2. Additional examples of biomarker tetrads that predict multiple sclerosis disease activity include: 1) NEFL, CCL20, SERPINA9, and TNFSF13B, 2) NEFL, APLP1, SERPINA9, and TNFSF13B, 3) NEFL, CCL20, APLP1, and TNFSF13B, 4) MOG, CXCL9, OPG, and TNFSF13B, 5) MOG, OPG, SERPINA9, and TNFSF13B, 6) MOG, CXCL9, SERPINA9, and TNFSF13B, and 7) CDCP1 and IL-12B.

[0130] In various embodiments, a minimal set of predictive biomarkers is useful for generating a prediction of MS disease progression. Examples of biomarker pairs useful for generating a prediction of MS disease progression include: 1) MOG and GFAP, 2) NEFL and GFAP, 3) GFAP and APLP1, and 4) NEFL and MOG. Examples of biomarker triplets useful for generating a prediction of MS disease progression include: 1) NEFL, MOG, and GFAP, 2) NEFL MOG, and GH, 3) NEFL, MOG, and SERPINA9, 4) OPN, CXCL13, and SERPINA9, 5) OPN, CXCL13, and TNFRSF10A, and 6) NEFL, CD6, and CXCL13. Examples of biomarker tetrads useful for generating predictions of MS disease progression include: 1) NEFL, MOG, GFAP, and IL-12B, 2) NEFL, MOG, GFAP, and PRTG, 3) NEFL, MOG, GFAP, and APLP1, 4) NEFL, MOG, SERPINA9, and GH, 5) NEFL MOG, TNFRSF10A, and SERPINA9, 6) CD6, CCL20, IL-12B, and APLP1, 7) CD6, CDCP1, IL-12B, and VCAN.

[0131] In various embodiments, at least one of the biomarkers in the minimal set of predictive biomarkers is GFAP. For example, at least one biomarker in a biomarker pair, triplet, or tetrad is GFAP. Examples of biomarker pairs that predict MS disease progression, one of which is GFAP, include: 1) GFAP and MOG, 2) GFAP and APLP1, 3) OPG and GFAP, 4) TNFRSF10A and GFAP, 5) GFAP and CDCP1, 6) GFAP and NEFL, 7) CNTN2 and GFAP, 8) GH and GFAP, and 9) CXCL9 and GFAP. Examples of biomarker triplets predicting MS disease progression in which one marker is GFAP include: 1) OPG, GFAP, and MOG; 2) OPG, GFAP, and APLP1; 3) GFAP, TNFRSF10A, and MOG; 4) CXCL9, OPG, and GFAP; 5) GFAP, TNFRSF10A, and APLP1; 6) GFAP, APLP1, and NEFL; 7) GFAP, CXCL13, and APLP1; 8) GFAP, FLRT2, and APLP1; 9) CXCL9, GFAP, and APLP1; and 10) GH, GFAP, and APLP1. Examples of biomarker tetrads predicting MS disease progression, one of which is GFAP, include: 1) CXCL9, OPG, GFAP, and MOG, 2) CNTN2, OPG, GFAP, and MOG, 3) CXCL9, OPG, GFAP, and APLP1, 4) OPG, GFAP, PRTG, and MOG, 5) OPG, GFAP, OPN, and MOG, 6) GFAP, CXCL13, APLP1, and NEFL, 7) GFAP, FLRT2, APLP1, and NEFL, 8) OPN, GFAP, APLP1, and NEFL, 9) CXCL9, GFAP, APLP1, and NEFL, and 10) GFAP, CXCL13, FLRT2, and APLP1. Pairs, triplets, or tetrads of these example biomarkers may be useful for predicting MS disease progression according to a conventional scale (e.g., EDSS or PDDS scoring scale).

[0132] Additional examples of biomarker pairs that predict MS disease progression, one of which is GFAP, include: 1) CDCP1 and GFAP, 2) APLP1 and GFAP, 3) GFAP and CXCL13, 4) MOG and GFAP, and 5) OPG and GFAP. Additional examples of biomarker triplets that predict MS disease progression, one of which is GFAP, include: 1) CDCP1, APLP1, and GFAP, 2) CDCP1, MOG, and GFAP, 3) APLP1, GFAP, and CXCL13, 4) CDCP1, GFAP, and SERPINA9, and 5) MOG, GFAP, and CXCL13. Additional examples of biomarker tetrads predictive of MS disease progression, one of which is GFAP, include: 1) CDCP1, CCL20, APLP1, and GFAP, 2) CDCP1, APLP1, GFAP, and CXCL13, 3) CDCP1, CCL20, MOG, and GFAP, 4) CDCP1, GFAP, APLP1, and SERPINA9, and 5) CDCP1, MOG, GFAP, and APLP1. These exemplary biomarker pairs, triplets, or tetrads may be useful in distinguishing between two classes of MS disease progression (e.g., mild / moderate and severe disability).

[0133] In various embodiments, the minimum set of predictive biomarkers need not include GFAP. Examples of pairs of biomarkers that predict MS disease progression, one of which is GFAP, include: 1) OPG and NEFL, 2) OPG and OPN, 3) OPG and FLRT2, 4) OPG and MOG, 5) CXCL9 and OPG, 6) GH and NEFL, 7) CXCL13 and NEFL, 8) APLP1 and NEFL, 9) CCL20 and NEFL, and 10) CXCL9 and NEFL. Examples of biomarker triplets predicting MS disease progression in which one biomarker is GFAP include: 1) OPG, MOG, and NEFL, 2) OPG, FLRT2, and NEFL, 3) CXCL9, OPG, and NEFL, 4) OPG, CDCP1, and NEFL, 5) OPG, OPN, and NEFL, 6) GH, APLP1, and NEFL, 7) GH, CXCL13, and NEFL, 8) GH, CDCP1, and NEFL, 9) CXCL13, CCL20, and NEFL, and 10) GH, CCL0, and NEFL. Examples of biomarker tetrads predicting MS disease progression, one of which is GFAP, include: 1) CDCP1, CXCL13, MOG, and NEFL, 2) CD6, CXCL9, CXCL13, and NEFL, 3) CXCL9, CXCL13, MOG, and NEFL, 4) CD6, CDCP1, CXCL13, and NEFL, 5) CD6, CXCL9, CXCL13, and MOG, 6) CDCP1, CXCL13, MOG, and NEFL, 7) CD6, CDCP1, CXCL13, and NEFL, 8) CXCL9, CXCL13, MOG, and NEFL, 9) CD6, CXCL9, CXCL13, and NEFL, and 10) CD6, CDCP1, CXCL13, and MOG. Pairs, triplets, or tetrads of these example biomarkers may be useful for predicting MS disease progression according to a conventional scale (e.g., EDSS or PDDS scoring scale).

[0134] V. Biomarkers Dysregulation of the biomarkers disclosed herein may contribute to the development and / or progression of disease activity, e.g., in neurodegenerative diseases, including multiple sclerosis, Parkinson's disease, Lewy body disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, Huntington's disease, spinal muscular atrophy, Friedreich's ataxia, Batten disease, and the like. Biomarkers and their corresponding classifications are shown below in Table 7. Exemplary categories include: neurodegeneration, myelin integrity, axonal integrity, cerebrovascular function, neurite outgrowth and neurogenesis, inflammation, neuroinflammation, immunomodulation, cell regulation, cell adhesion, brain-gut axis, metabolism, and neuromodulation. Exemplary biomarker classifications are shown in Figure 1D. Additionally, biomarkers and their involvement in specific locations (e.g., brain, brain barrier, or blood) and cell types are shown in Tables 8, 9A, and 9B.

[0135] NEFL is a 68 kDa biomarker that reflects axonal damage in the microenvironment. In other words, NEFL often serves as a surrogate for axonal degeneration. Furthermore, NEFL interacts with other biomarkers, such as MAP2, protein kinase N1, and tuberous sclerosis complex (TSC1).

[0136] COL4A1 is a 26-kDa biomarker involved in cell proliferation, migration, extracellular matrix formation, and the inhibition of endothelial cell proliferation, migration, and tube formation. COL4A1 is involved in the outgrowth of hippocampal embryonic neurons and in the integrity of myelin. Type IV collagen is a major component of the glomerular basement membrane (GBM) and forms a wire meshwork with laminin, proteoglycans, and entactin / nidogen. It contains a C-terminal NC1 domain that inhibits angiogenesis and tumorigenesis. The C-terminal half has been shown to have antiangiogenic activity. Type IV collagen also inhibits endothelial cell proliferation, migration, and tube formation, as well as the expression of hypoxia-inducible factor 1α, ERK1 / 2, and p38 MAPK activation. COL4A1 mutations are associated with a wide range of phenotypes, including both ischemic and hemorrhagic stroke, migraine, leukomalacia, nephropathy, hematuria, chronic muscle spasms, and anterior segment eye disorders, including congenital cataracts, glaucoma, and Axenfeld-Rieger syndrome. Case Rep Neurol. 2015 May-Aug;7(2):142-147. Published online 2015 Jun 2. doi:10.1159 / 000431309.

[0137] APLP1 is a 72-kDa biomarker involved in regulating synapse maturation and neurite outgrowth during cortical development. APLP1 is one of two homologs of amyloid-like proteins 1 and 2, or APLP1 and APLP2. The gene encoding APLP1 is a member of the highly conserved amyloid precursor protein gene family. The encoded protein is a membrane-bound glycoprotein that is cleaved by secretases in a manner similar to the cleavage of the amyloid-βA4 precursor protein. This cleavage releases an intracellular cytoplasmic fragment that can act as a transcriptional activator. APLP1 may also be involved in synapse maturation during cortical development. It may regulate neurite outgrowth by binding to components of the extracellular matrix, such as heparin and collagen I. APLP1 is widely expressed in humans. Functions attributed to APLP1 include neurite outgrowth and synaptogenesis, protein transport along axons, cell adhesion, calcium metabolism, neuronal injury, synaptic dysfunction, and signal transduction.

[0138] MMP-2 (72 kDa) and MMP-9 (78-92 kDa) are gelatinases, a type of proteolytic enzyme involved in the degradation of the extracellular matrix. MMP-2 and MMP-9 are involved in physiological processes such as embryonic development, reproduction, and tissue remodeling. Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes. Avolio, C., et al. Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes, J. Neuroimmunol. Mar;136(1-2):46-53. The integrity of the blood-brain barrier, as the major structural interface between the periphery and the brain, appears to play an important role in MS. Reducing the secretion of proteolytic matrix metalloproteinases (MMPs), such as MMP-2 and / or MMP-9, as disruptors of blood-brain barrier integrity, may have a significant impact on MS. Proschinger et al. “Influence of combined functional resistance and exercise endurance over 12 weeks on matrix metalloproteinase-2 serum concentration in persons with relapsing-remitting multiple sclerosis-a community-based randomized controlled trial.” BMC Neurol 19,314 (2019).

[0139] FLRT2 is a 74 kDa biomarker and a member of the fibronectin leucine-rich transmembrane protein family that functions in cell adhesion and / or receptor signaling. FLRT2 is also expressed in the brain, heart, and several other organs, where it is involved in fibroblast growth factor-mediated signaling cascades. In the heart, it is required for the normal organization of the cardiac basement membrane during embryogenesis, as well as for normal embryonic epicardial and cardiac morphogenesis. In the context of neurology, FLRT2 functions in cell-cell adhesion, cell migration, and axon guidance. It may be involved in cortical neuron migration during brain development through its interaction with UNC5D. FLRT2 is also involved in glutamate excitotoxicity, neuronal cell death, and synaptogenesis and plasticity.

[0140] VCAN (a >200 kDa biomarker) is involved in cell motility, cell growth and differentiation, cell adhesion, cell proliferation, cell migration, and angiogenesis. VCAN is also involved in myelin protection, astrocytic excitotoxicity, and secreted inflammatory mediators. VCAN is a key factor in inflammation through its interaction with adhesion molecules on the surface of inflammatory leukocytes and with chemokines involved in the recruitment of inflammatory cells. In the adult central nervous system, versican is found in the perineural network, where it may stabilize synaptic connections. Versican may also inhibit nervous system regeneration and axonal growth after central nervous system injury.

[0141] TNFSF13B, also referred to herein as B cell-activating factor (BAFF), is a biomarker involved in T cell-independent B cell activation and ectopic lymphoid follicle formation.

[0142] CHI3L1 is a 40-kDa biomarker involved in inflammation, the innate immune system, tissue remodeling, and the ability of cells to respond to and cope with environmental changes. Furthermore, CHI3L1 is involved in T helper cell type 2 (Th2) inflammatory responses and IL-13-induced inflammation, regulating allergen sensitization, inflammatory cell apoptosis, dendritic cell accumulation, and M2 macrophage differentiation. CHI3L1 promotes the invasion of pathogenic enterobacteria into the colonic mucosa and lymphoid organs by contributing to macrophage bacterial killing, controlling bacterial dissemination, and enhancing host resistance, activating the AKT1 signaling pathway in colonic epithelial cells and subsequent IL-8 production, and promoting antibacterial responses in the lungs. CHI3L1 also regulates hyperoxia-induced lung injury, inflammation, and epithelial apoptosis.

[0143] IL-12B is a 40 kDa biomarker that represents one subunit of the IL-12 heterodimer. IL-12A (35 kDa) represents the other subunit of the IL-12 heterodimer. IL-12B is involved in innate and adaptive immunity and the regulation of memory / effector Th1 cells. IL-12B is a growth factor for activated T cells and NK cells. IL-12B associates with IL-23A to form the IL-23 interleukin, a heterodimeric cytokine that functions in innate and adaptive immunity. Polymorphisms in the genes encoding the interleukin-23 receptor (IL23R) and the p40 subunit of IL-12 / 23 (IL12B) are associated with the risk of multiple sclerosis (MS). Huang et al., “Meta-analysis of the IL23R and IL12B polymorphisms in multiple sclerosis.” Int.J.of Neuroscience, 126:3, 205-212 (2016).

[0144] IFI30 is a 30-35 kDa biomarker involved in antigen processing by promoting the complete unfolding of proteins destined for lysosomal degradation. IFI30 promotes the generation of MHC class II-restricted epitopes from disulfide-bond-containing antigens through endocytic reduction of disulfide bonds. IFI30 also promotes MHC class I-restricted recognition of exogenous antigens containing disulfide bonds by CD8+ T cells or cross-presentation. IFI30 is constitutively expressed in antigen-presenting cells and is induced by inflammatory cytokines.

[0145] SERPINA9 is a 42 kDa biomarker that is a member of the serpin family of serine protease inhibitors. SERPINA9 is involved in neuronal injury. SERPINA9 expression may be restricted to germinal center B-cell and lymphoid malignancies. SERPINA9 may function in vivo in germinal centers as an efficient inhibitor of trypsin-like proteases.

[0146] IL-18 is involved in immune responses and inflammatory processes. IL-18 is a proinflammatory cytokine primarily involved in polarized T helper 1 (Th1) and natural killer (NK) cell immune responses. It serves as an inhibitor of early Th1 cytokine responses. Furthermore, it contributes to Th-1 responses through its ability to induce IFN-γ production in T cells and NK cells. IL-18 levels in CSF and serum were significantly higher than those found in patients without lesions. These results suggest the involvement of IL-18 in the immunopathogenesis of MS, especially during the active phase of the disease. (Losy, J., et al., IL-18 in patients with multiple sclerosis. Acta Neurologica Scandinavica, 104:171-173 (2001)). Furthermore, higher IL-18 serum levels and significantly different frequencies of two IL-18 polymorphisms were observed in MS patients. Jahanbani-Ardakani, H. et al., Interleukin 18 polymorphisms and its serum level in patients with multiple sclerosis, Ann Indian Acad Neurol;22:474-76(2019).

[0147] CDCP1 is a 90-140 kDa biomarker involved in T cell migration, cell adhesion, and cell-matrix association. Through phosphorylation, CDCP1 may be involved in regulating anchorage versus migration or proliferation versus differentiation. CDCP1 is expressed in cells with phenotypes reminiscent of mesenchymal stem cells and neural stem cells. Furthermore, CDCP1 is a ligand for CD6, a receptor molecule expressed on certain T cells, and may be involved in migration and chemotaxis.

[0148] CNTN2 is a 113-kDa biomarker involved in cell adhesion, proliferation, migration, neuronal axon guidance, neuronal injury, and axon-dendritic rearrangement. CNTN2 is a member of the contactin family of proteins, part of the immunoglobulin superfamily of cell adhesion molecules. CNTN2 is a glycosylphosphatidylinositol (GPI)-anchored neuronal membrane protein involved in the proliferation, migration, and axon guidance of neurons in the developing cerebellum. Mutations in the CNTN2 gene can be associated with adult myoclonic epilepsy. Together with another transmembrane protein, CNTNAP2 contributes to the organization of the axonal domains at the nodes of Ranvier by maintaining voltage-gated potassium channels in the paranodal region.

[0149] GFAP is a 50kDa biomarker involved in demyelination, degeneration, and axonal injury. Astroglial activation, associated with immune cascade activation, is thought to contribute to the demyelination and axonal injury observed in MS. Glial fibrillary acidic protein (GFAP) is a major component of the glial scar. GFAP is used as a marker to distinguish astrocytes from other glial cells during development. Higher serum concentrations of both GFAP and NEFL were associated with higher EDSS, older age, longer disease duration, progressive disease course, and MRI pathology. Hoegel, H., et al. Serum glial fibrillary acidic protein correlates with multiple sclerosis disease severity. Multiple Sclerosis Journal, 26(13)2018.

[0150] MOG is a 28-kDa membrane protein expressed on the oligodendrocyte cell surface and the outermost surface of the myelin sheath. Due to this localization, it serves as a cell surface receptor or cell adhesion molecule, making it a major target antigen involved in immune-mediated demyelination. This protein may be involved in the completion and maintenance of the myelin sheath and intercellular communication. Diseases associated with MOG include narcolepsy and rubella. Related pathways include neural stem cell differentiation pathways and lineage-specific markers. A paralog of the MOG gene is BTN1A1.

[0151] CD6 is a 90-130 kDa biomarker involved in central nervous system development. CD6 is a cell adhesion molecule involved in blood-brain barrier breach and T cell-mediated acute inflammatory responses. Recent studies have identified CD6 as a risk gene for multiple sclerosis (MS), a disease in which autoreactive T cells are integrally involved. De Jager, PL., et al., Meta-analysis of genome scans and replication identify CD6, IRF8, and TNFRSF1A as new multiple sclerosis susceptibility loci. Nat Genet. 2009;41(7):776-782. CD6 is found on the outer membrane of T lymphocytes and is involved in leukocyte migration across the blood-brain barrier.

[0152] CXCL9 is a 12-kDa biomarker involved in immune responses and inflammatory processes. CXCL9 is a cytokine that affects the growth, migration, or activation state of cells involved in immune and inflammatory responses. CXCL9 (MIG) is a chemokine that, upon binding to the receptor CXCR3, induces chemotactic activity for T cells and is involved in inflammatory responses. CXCL9 is not constitutively expressed but is induced by IFN-γ. CXCL9 has been implicated in several inflammation-related diseases, such as hepatitis C, skin inflammation, rheumatoid arthritis, and pharyngitis. Consistent with this observation is the upregulation of the ELR-CXC chemokines CXCL9, CXCL10, and CXCL11 in the CNS of mice with EAE, which is induced by Th1 cell migration. Lovett-Racke, A. et al. Th1 versus Th17:Are T cell cytokines relevant in multiple sclerosis?Biochimca et Biophysica Acta(BBA)-Molecular Basis of Disease.1812(2):246-251(2011).

[0153] CXCL13 is a biomarker involved in cell proliferation, cell regeneration, regeneration, and inflammatory responses. CXCL13 belongs to the CXC chemokine family and is selectively chemotactic for B cells. It interacts with the chemokine receptor CXCR5, which regulates B cell organization. Serum levels of CXCL13 are involved in multiple sclerosis. Festa, E. et al. Serum levels of CXCL13 are elevated in active multiple sclerosis. Multiple Sclerosis Journal, 15(11):1271-1279 (2009).

[0154] CCL20 is an 11 kDa biomarker involved in axon guidance and chemotaxis of dendritic cells. CCL20 is a chemokine involved in immunoregulation and inflammatory processes (e.g., acute inflammatory responses) and is expressed in epithelial cells of the choroid plexus of the human brain. CCL20 serves as the cognate ligand for CCR6.

[0155] OPG is a 55-60 kDa biomarker involved in inflammation, cell apoptosis, and T cell activation processes. OPG is a decoy receptor for the cytokines TNFSF11 (RANKL) and possibly TNFSF10 (TRAIL) and belongs to the TNF receptor superfamily. OPG is upregulated by estrogen and increasing calcium concentrations and is involved in inflammation, innate immunity, and transcriptional regulation in cell survival and differentiation. For example, OPG binding to TNFSF11 inhibits the differentiation of osteoclast precursor cells into mature osteoclasts, and OPG has been used experimentally to treat osteoporosis. OPG has been implicated in several inflammation-related diseases, such as rheumatoid arthritis, inflammatory bowel disease, and periodontitis.

[0156] OPN is a 33-44 kDa biomarker involved in inflammation and immune regulation. OPN is a pleiotropic integrin-binding protein that functions in cell-mediated immunity, inflammation, tissue repair, and cell survival. OPN is also involved in biomineralization.

[0157] PRTG is a 180 kDa biomarker involved in neurogenesis, neurotrophin binding, neuronal survival, and demyelination. It may be involved in anterior-posterior axis elongation. PRTG is a membrane protein and a member of the immunoglobulin superfamily. It is primarily thought to be a developmental protein with some relevance to neuralgia, demyelinating diseases, and dyslexia.

[0158] TNFRSF10A is a 50 kDa biomarker that is a member of the TNF receptor superfamily. TNFRSF10A is involved in inflammatory and neurodegenerative processes. This receptor is activated by tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10 / TRAIL), thereby transmitting a cell death signal and inducing cell apoptosis.

[0159] GH, also known as somatotropin or somatropin, is a neuroendocrine marker that stimulates growth, cell proliferation, and regeneration in humans and other animals. GH regulates energy homeostasis and metabolism. GH is a mitogen specific to certain cell types. Previous studies have shown that it is decreased in the serum of patients with severe MS. Gironi, M., et al. Growth hormone and disease severity in early stage of multiple sclerosis. Multiple Sclerosis International 2013:(2013).

[0160] GH2, also known as growth hormone 2, placenta-specific growth hormone, and growth hormone variant, is a biomarker involved in the growth control, differentiation, and proliferation of myoblasts. It regulates energy homeostasis and metabolism. It is produced and secreted by the placenta during pregnancy and is the major form of growth hormone during pregnancy.

[0161] VCAM-1 is an 80 kDa transmembrane biomarker normally expressed on blood vessels and mediates cell adhesion to the vascular endothelium. VCAM-1 is characterized by multiple immunoglobulin domains. VCAM-1 has been implicated in multiple sclerosis. Peterson, J. et al., VCAM-1-Positive Microglia Target Oligodendrocytes at the Border of Multiple Sclerosis Lesions, Journal of Neuropathology & Experimental Neurology, Volume 61, Issue 6, June 2002, Pages 539-546. Matsuda, M. et al. Increased levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in the cerebrospinal fluid and serum of patients with multiple sclerosis and human T lymphotropic virus type-1-associated myelopathy. J. Neuroimmunology, 59(1-2):35-40 (1995).

[0162] In various embodiments, the biomarker panel may further comprise additional biomarkers as described herein. In various embodiments, these additional biomarkers serve as surrogate biomarkers for the biomarkers described above. In various embodiments, the additional biomarkers include: cell adhesion molecule 3 (CADM3), kallikrein-related peptidase 6 (KLK6), brevican (BCAN), oligodendrocyte myelin glycoprotein (OMG), CD5 molecule (CD5), cytotoxic and regulatory T cell molecule (CRTAM), CD244 molecule (CD244), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), proteinase 3 (PRTN3), follistatin-like 3 (FST), and / or IFN-γ receptor 4 (IFN-γ receptor 4). L3), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 11 (CXCL11), interleukin-18 binding protein (IL-18BP), macrophage scavenger receptor 1 (MSR1), C-C motif chemokine ligand 3 (CCL3), tumor necrosis factor ligand superfamily member 12 (TWEAK), trefoil factor 3 (TFF3), ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2) ), insulin-like growth factor binding protein 1 (IGFBP-1), interleukin 12A (IL12A), seizure-related 6 homolog-like (SEZ6L), dipeptidyl peptidase-like 6 (DPP6), neurocan (NCAN), tubulointerstitial nephritis antigen-like 1 (TINAGL1), calcium-activated nucleotidase 1 (CANT1), nectin cell adhesion molecule 2 (NECTIN2), neural proliferation, differentiation and regulation protein 1 (NPDC1), tumor necrosis factor receptor superfamily member 1 1A (TNFRSF11A), contactin 4 (CNTN4), neurotrophic receptor tyrosine kinase 2 (NTRK2), neurotrophic receptor tyrosine kinase 3 (NTRK3), cadherin 6 (CDH6), carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8), mitotic arrest deficient 1-like 1 (MAD1L1), Fc fragment of IgA receptor (FCAR), myeloperoxidase (MPO), osteomodulin (OMD), matrix extracellular phosphoglycoprotein (MEPE),GDNF family receptor α3 (GDNFR-α3), scavenger receptor class F member 2 (SCARF2), CD40 ligand (IgM), tumor necrosis factor receptor superfamily member 1B (TNF-R2), programmed cell death 1 ligand (PD-L1), Notch3 (NOTCH3), contactin 1 (CNTN1), oncostatin M (OSM), transforming growth factor α (TGF-α), peptidoglycan recognition protein 1 (PGLYRP1), nitric oxide synthase 3 (NOS3), discoidin domain receptor tyrosine kinase 1 (DDR1), C-X-C motif chemokine ligand 16 (CXCL16), CD166 antigen (ALCAM), spondin 2 (SPON2), and protocadherin 17 (PCDH17).

[0163] CADM3 is involved in cell-cell adhesion and interacts with IGSF4, NECTIN1, NECTIN3, and EPB41L1. CADM3 is involved in the biological processes of adherens junction organization, heterophilic cell-cell adhesion, homophilic cell adhesion, and protein localization.

[0164] KLK6 is a serine protease that exhibits activity against proteins such as α-synuclein, amyloid precursor protein, myelin basic protein, gelatin, casein, and extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen. KLK6 is involved in the biological processes of amyloid precursor protein metabolism, CNS development, myelination, protein autoprocessing, regulation of cell differentiation and neuronal development, wound response, and tissue regeneration.

[0165] BCAN is a proteoglycan and a member of the lectican protein family. BCAN is involved in the biological processes of cell adhesion, CNS development, chondroitin sulfate biosynthesis and catabolic processes, extracellular matrix organization, and axonal synapse maturation.

[0166] OMG is a cell adhesion molecule involved in myelination of the central nervous system. OMG is involved in the biological processes of cell adhesion, regulation of axon formation, and neuronal projection regeneration.

[0167] CD5 is a signaling molecule that can be expressed on the surface of cells such as T lymphocytes. CD5 is involved in the biological processes of apoptosis signaling pathways, cell recognition, T cell proliferation, and T cell costimulation.

[0168] CRTAM is an immunoglobulin superfamily transmembrane protein that is involved in the biological processes of adaptive immune response, cell recognition, stimuli or cell detection, and regulation of immune responses. CRTAM is also involved in heterophilic cell-cell adhesion, which regulates the activation, differentiation, and tissue retention of various T cell subsets.

[0169] CD244 is a member of the signaling lymphocyte activation molecule family expressed on natural killer cells. CD244 is involved in the biological processes of immune responses (adaptive and innate), leukocyte migration, regulation of cytokine secretion, and signal transduction. CD244 regulates the activation and differentiation of a wide variety of immune cells, thereby participating in the regulation and interconnection of both innate and adaptive immune responses.

[0170] TNFRSF9 is a member of the tumor necrosis factor receptor family and is involved in the biological processes of TNFR signaling pathway, cell proliferation, and apoptosis. TNFRSF9 is expressed by activated T cells.

[0171] PRTN3 is a serine protease expressed by neutrophil granulocytes and is involved in the biological processes of antibacterial humoral responses, blood coagulation, neutrophil activity, proteolysis, and cytokine-mediated signaling pathways.

[0172] FSTL3 is a secreted glycoprotein of the follistatin module protein family. FSTL3 is involved in the biological processes of activin / fibronectin binding, organ development, bone formation, ossification, and regulation of cell-cell adhesion.

[0173] CXCL10 and CXCL11 are cytokines of the CXC chemokine family, respectively, that are involved in the biological processes of immune response, inflammatory response, cell signaling, chemotaxis, T cell recruitment, and cell proliferation.

[0174] IL-18BP is a protein that serves as an inhibitor of the proinflammatory cytokine IL18. IL-18BP is involved in the biological processes of cytokine stimulation, IL-18-mediated signaling pathways, and immune responses.

[0175] MSR1 is a membrane glycoprotein expressed by macrophages. MSR1 is involved in the biological processes of endocytosis and cholesterol transport and storage, and may be involved in the pathological deposition of cholesterol in arterial walls during atherogenesis.

[0176] CCL3 is a monokine with inflammatory and chemokinetic properties that binds to CCR1, CCR4, and CCR5. CCL3 is involved in the biological processes of cell migration (e.g., lymphocyte and macrophage chemotaxis), calcium-mediated signaling, intercellular signaling, cytokine secretion, and inflammatory responses.

[0177] TWEAK is a cytokine of the tumor necrosis factor ligand family. TWEAK is involved in the biological processes of angiogenesis, cell differentiation, immune response, signal transduction, apoptosis, and TNF-mediated signal transduction pathways. TWEAK also promotes endothelial cell proliferation and migration.

[0178] TFF3 is a 6 kDa glycoprotein normally produced by goblet cells and involved in the gastrointestinal tract. TFF3 is involved in the biological processes of maintaining and healing the gastrointestinal epithelium and regulating glucose metabolic processes.

[0179] ENPP2 is a phosphodiesterase involved in the production of lysophosphatidic acid, a lipid signaling molecule that hydrolyzes lysophospholipids and is involved in the biological processes of cell motility, chemotaxis, immune response, and angiogenesis.

[0180] IGFBP-1 is a member of the insulin-like growth factor binding protein family. It binds to insulin-like growth factors (IGF) I and II. IGFBP-1 is involved in the biological processes of aging, cellular metabolic processes, cell growth, signal transduction, and tissue regeneration.

[0181] IL-12A is a subunit that, together with the other IL-12B subunit, forms the IL-12 heterodimer. IL-12A is involved in the biological processes of cell migration, cell proliferation, cell adhesion, cell differentiation, and regulating immune cell (e.g., T cells, dendritic cells, and natural killer cells) activation.

[0182] SEZ6L is a protein located primarily within the endoplasmic reticulum membrane, which regulates endoplasmic reticulum function in neurons. SEZ6L is involved in the biological processes of synapse maturation, adult motor behavior, and regulation of protein kinase C signaling.

[0183] DPP6 is a membrane protein that is a member of the peptidase S9B family of serine proteases. DPP6 is involved in the biological processes of potassium channel regulation and protein localization to the plasma membrane, and may influence susceptibility to amyotrophic lateral sclerosis.

[0184] NCAN is a protein that is a member of the lectican / chondroitin sulfate proteoglycan family. NCAN is involved in the biological processes of cell adhesion, CNS development, ECM organization, and chondroitin sulfate and dermatan synthesis. NCAN is involved in neural cell adhesion and neurite outgrowth during development by binding to neural cell adhesion molecules.

[0185] TINAGL1 is an extracellular matrix protein involved in the biological processes of cell adhesion, proliferation, migration, and differentiation. TINAGL1 is also involved in endocytosis and endosomal trafficking.

[0186] CANT1 is a calcium-dependent nucleotidase with a preference for uridine diphosphate. CANT1 is involved in the biological processes of regulating calcium ion binding, neutrophil degranulation, NF-κB signaling, and proteoglycan biosynthesis. CANT1 regulates metabolic processes such as nucleotide metabolism.

[0187] Nectin2 is a membrane glycoprotein that serves as a component of adherens junctions and is involved in the biological processes of cell-cell adhesion (via adherens junctions), cytoskeletal organization, viral receptor activity, and regulation of NK and T cell activity.

[0188] NPDC1 is a protein primarily expressed in the brain. NPDC1 is involved in the biological processes of immune response regulation and neuronal development and proliferation. NPDC1 suppresses oncogenic transformation of neuronal and non-neuronal cells and downregulates neuronal proliferation.

[0189] TNFRSF11A is a member of the TNF receptor superfamily. TNFRSF11A is involved in the biological processes of cell-cell signaling, immune response, monocyte chemotaxis, and TNF-mediated signaling pathways. Furthermore, TNFSF11A is involved in osteoclastogenesis.

[0190] CNTN4 is a member of the contactin family of immunoglobulins. CNTN4 is involved in the biological processes of axon guidance and development, synaptogenesis, cell-surface interactions during nervous system development, brain development, neuron-cell adhesion, neuronal projection, neuronal differentiation, and regulation of synaptic plasticity.

[0191] NTKR2 is a receptor tyrosine kinase (part of the neurotrophic factor tyrosine receptor kinase family) that binds to brain-derived neurotrophic factors and is involved in the biological processes of neuronal survival, proliferation, migration, differentiation, synaptogenesis, and synaptic plasticity.

[0192] NTKR3 is a receptor tyrosine kinase (part of the neurotrophin tyrosine receptor kinase family) that binds to neurotrophin 3. NTKR3 is involved in the biological processes of regulating GTPase and MAPK activity, astrocyte differentiation, nervous system development, and neuronal migration.

[0193] CDH6 is a member of the cadherin superfamily that mediates cell-cell adhesion. CDH6 is involved in the biological processes of cell-cell adhesion (adherens junctions), cell morphogenesis, and the Notch signaling pathway. CDH6 mediates both heterotypic and homotypic cell-cell contacts through its interaction with CD6. CDH6 is also involved in axon outgrowth and axon guidance.

[0194] CEACAM8 is a cell surface glycoprotein that belongs to the carcinoembryonic antigen (CEA) superfamily. CEACAM8 is involved in the biological processes of regulating immune responses, leukocyte migration, neutrophil degranulation, and cell-cell adhesion.

[0195] MAD1L1 is a mitotic spindle assembly checkpoint protein that is involved in the biological processes of cell division and mitotic cell cycle checkpoints.

[0196] FCAR is a transmembrane glycoprotein on the surface of immune cells such as neutrophils, monocytes, and macrophages. FCAR is involved in the biological processes of regulating immune responses, neutrophil activation / degranulation, and response to cytokines (e.g., interferons, interleukins, TNF).

[0197] MPO is a hemoprotein (enzyme) expressed in neutrophil granulocytes. MPO is involved in the biological processes of immune response, neutrophil degranulation, and chromatin / heme / heparin binding.

[0198] OMD is suggested to be involved in the biomineralization process and osteoblast attachment. OMD is involved in regulating the biological processes of cell adhesion and bone mineralization.

[0199] MEPE is a calcium-binding phosphoprotein of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family. MEPE is involved in the biological processes of extracellular matrix binding / regulation, biomineralization, skeletal development, and bone and cartilage mineralization. MEPE is involved in renal phosphate excretion and inhibits intestinal phosphate absorption. MEPE is also involved in the proliferation and differentiation of dental pulp stem cells.

[0200] GDNFR-α3 is a glial cell line-derived neurotrophic factor (GDNF) receptor family member that binds to artemin (ARTN). GDNFR-α3 is involved in the biological processes of axon guidance, nervous system development, neuronal migration, GDNF receptor activity, and signaling receptor activity and binding.

[0201] SCARF2 is a member of the scavenger receptor type F family. SCARF2 is an adhesion protein and is involved in the biological processes of scavenger receptor activity and cell-cell adhesion.

[0202] CD40 ligand is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. CD40 ligand is involved in the biological processes of B cell differentiation and proliferation, inflammatory responses, leukocyte intercellular adhesion, platelet activation, T cell costimulation, and TNF-mediated signaling pathways.

[0203] TNF-R2 is a membrane receptor that binds to tumor necrosis factor alpha. TNF-R2 is involved in the biological processes of TNF-mediated signaling pathways, neutrophil degranulation, regulation of neuroinflammatory responses, and cell signaling. TNF-R2 protects neurons from apoptosis by stimulating antioxidant pathways.

[0204] PD-L1 is a ligand that binds to PD-1 and is an important target in research into checkpoint inhibitors for cancer immunotherapy. PD-L1 is involved in the biological processes of immune response, interferon regulation, T cell proliferation, T cell costimulation, cell migration, and cytokine production.

[0205] NOTCH3 is a member of the NOTCH receptor family of proteins involved in the Notch signaling pathway. NOTCH3 is involved in the biological processes of gene activation, calcium ion binding, signaling receptor activity, neuronal fate determination, and brain development. Furthermore, NOTCH3 regulates cell fate determination.

[0206] CNTN1 is a neuronal membrane protein involved in cell adhesion. CNTN1 is involved in the biological processes of neuronal projection development, brain development, cell adhesion, and Notch signaling.

[0207] OSM is a cytokine of the interleukin 6 cytokine family. OSM is involved in the biological processes of immune response, cell growth / division, inflammatory response, and regulation of cytokine activity (e.g., MAPK and STAT pathways).

[0208] TGFA is a mitogenic polypeptide and part of the epidermal growth factor family. TGFA is involved in the biological processes of growth factor activity, signaling pathways (e.g., MAPK, EGF), cell division / proliferation, and signal transduction.

[0209] PGLYRP1 is a peptidoglycan-binding protein that is involved in the biological processes of innate immune responses, inflammatory responses, neutrophil degranulation, and peptidoglycan immunoreceptor activity.

[0210] NOS3 regulates the production of nitric oxide, which is involved in the biological processes of angiogenesis, vascular remodeling, endothelial cell migration, vasodilation, vascular smooth muscle relaxation, and platelet activation promoting blood clotting.

[0211] DDR1 regulates cell adhesion to the extracellular matrix, extracellular matrix remodeling, cell migration, differentiation, and survival and proliferation. Furthermore, DDR1 promotes smooth muscle cell migration.

[0212] CXCL166 is involved in immune regulation and serves as a scavenger receptor for macrophages.

[0213] IL6 is a cytokine involved in the differentiation of B cells, lymphocytes, and monocytes.

[0214] ALCAM is involved in axon guidance, differentiation pathways of embryonic and induced pluripotent stem cells, and lineage-specific markers, as well as L1CAM interactions.

[0215] NTRK2 is involved in the development and maturation of the central and peripheral nervous systems through the regulation of neuronal survival, proliferation, migration, differentiation, and synaptogenesis and plasticity.

[0216] SPON2 is involved in the outgrowth of embryonic hippocampal neurons.

[0217] NTRK3 is involved in nervous system regulation and cardiac development.

[0218] PCDH17 is involved in the establishment and function of specific cell-cell junctions in the brain.

[0219] VI. Assay As shown in FIG. 1A, the system environment 100 includes a marker quantification assay 120 for assessing the expression level of one or more biomarkers. Examples of assays for one or more markers (e.g., the marker quantification assay 120) include DNA assays, microarrays, polymerase chain reaction (PCR), RT-PCR, Southern blots, Northern blots, antibody-binding assays, enzyme-linked immunosorbent assays (ELISAs), flow cytometry, protein assays, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, immunoassays, including but not limited to, RIAs, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, or competitive immunoassays, immunoprecipitation, and assays described in the Examples section below. Assay information can be quantitative and sent to the computer system of the present invention. Information can also be qualitative, such as observed patterns or fluorescence, which can be converted into quantitative measurements by the user or automatically by a reader or computer system.

[0220] Various immunoassays designed to quantify markers can be used in screening, including multiplex assays. Measuring the concentration of target markers in a sample or fraction thereof can be achieved by a variety of specific assays. For example, traditional sandwich-type assays can be used in formats such as arrays, ELISAs, and RIAs. Other immunoassays include Ouchterlony plates, which provide a simple measurement of antibody binding. Additionally, Western blots can be performed on protein gels or protein spots on filters using convenient labeling methods, optionally with marker-specific detection systems.

[0221] Protein-based assays using antibodies that specifically bind to polypeptides (e.g., markers) can be used to quantify marker levels in test samples obtained from subjects. In various embodiments, the antibodies that bind to markers can be monoclonal antibodies. In various embodiments, the antibodies that bind to markers can be polyclonal antibodies. For multiplexed analysis of markers, arrays containing one or more marker affinity reagents, e.g., antibodies, can be generated. Such arrays can be constructed that include antibodies against markers. Detection can utilize one or a panel of marker affinity reagents, for example, a panel or cocktail of affinity reagents specific for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more markers.

[0222] In various embodiments, multiplex assays involve the use of oligonucleotide-labeled antibody probes that bind to target biomarkers and allow subsequent quantification of the biomarkers. One example of a multiplex assay involving oligonucleotide-labeled antibody probes is proximity extension assay (PEA) technology (Olink Proteomics). Briefly, pairs of oligonucleotide-labeled antibodies bind to biomarkers, and the two oligonucleotide sequences are complementary to each other. Thus, the oligonucleotide sequences will hybridize to each other only if both antibodies bind to the target biomarkers. Mismatched oligonucleotide sequences (resulting from nonspecific antibody binding or antibody cross-reactivity) will not hybridize and therefore will not result in a readout. The hybridized oligonucleotide sequences undergo nucleic acid extension and amplification and are then quantified using microfluidic qPCR. The quantified levels correlate with the quantitative expression values ​​of each biomarker.

[0223] In various embodiments, multiplex assays involve the use of bead-conjugated antibodies (e.g., capture antibodies) that allow for the binding and detection of biomarkers. One example of a multiplex assay involving bead-conjugated antibodies is Luminex's xMAP® technology. Here, a bead-bound antibody is added to a sample along with a biotinylated detection antibody. Both antibodies are specific for the biomarkers of interest, thus forming an antibody-antigen sandwich. Streptavidin is then added, which binds to the biotinylated detection antibody and allows for the detection of the complex. A Luminex 200™ or FlexMap® analyzer is used to identify and quantify the amount of biomarkers in the sample. In various embodiments, multiplex assays represent an improvement over Luminex's xMAP® technology, e.g., the Multi-Analyte Profile (MAP) technology by Myriad Rules Based Medicine (RBM), Inc.

[0224] In various embodiments, a sample obtained from a subject may be processed prior to implementation of a marker quantification assay 120 (e.g., an immunoassay). In various embodiments, processing the sample enables implementation of a marker quantification assay 120 to more accurately assess the expression level of one or more biomarkers in the sample.

[0225] In various embodiments, a sample from a subject can be processed to extract biomarkers from the sample. In one embodiment, the sample can undergo phase separation to separate the biomarkers from other portions of the sample. For example, the sample can undergo centrifugation (e.g., pelleting or density gradient centrifugation) to separate larger and / or denser entities in the sample (e.g., cells and other macromolecules) from the biomarkers. Another example includes filtration (e.g., ultrafiltration) to phase separate the biomarkers from other portions of the sample.

[0226] In various embodiments, a sample from a subject can be processed to generate subsamples containing a fraction of the biomarkers present in the sample. In various embodiments, generating a fraction of the biomarkers can include performing a protein fractionation procedure. Examples of protein fractionation procedures include chromatography (e.g., gel filtration, ion exchange, hydrophobic chromatography, or affinity chromatography). In certain embodiments, the protein fractionation procedure includes affinity purification or immunoprecipitation, in which the biomarkers are bound by specific antibodies. Such antibodies can be immobilized on a support, such as a magnetic particle or nanoparticle or a plate.

[0227] In various embodiments, a sample from a subject is processed to extract biomarkers from the sample and further processed to generate a subsample containing a fraction of the extracted biomarker. Overall, this allows for a purified subsample of a particular biomarker of interest. Accordingly, implementing an assay (e.g., an immunoassay) to assess the expression level of a particular biomarker of interest can be more accurate and of higher quality. In various embodiments, the particular biomarker can be a biomarker of a biomarker panel, the embodiments of which are described herein. By way of example, the biomarkers of the biomarker panel can include two or more of NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20, IL-12B, APLP1, TNFRSFlOA, COL4A1, SERPINA9, FLRT2, TNFSF13B, GH, VCAN, PRTG, and CNTN2.

[0228] VII. THERAPEUTIC AGENTS AND COMPOSITIONS FOR THERAPEUTIC AGENTS In various embodiments, a therapeutic agent is provided to an individual before and / or after obtaining a sample from the individual and determining quantitative expression values ​​of one or more markers in the obtained sample. In one example, a predictive model receiving the quantitative expression values ​​predicts that an individual will be diagnosed with multiple sclerosis and that a therapeutic agent will be provided. In another example, the predictive model predicts that the provided therapeutic agent will demonstrate therapeutic efficacy for multiple sclerosis in a previously diagnosed individual.

[0229] In various embodiments, the therapeutic agent is a biologic, e.g., a cytokine, antibody, soluble cytokine receptor, antisense oligonucleotide, siRNA, etc. Such biologic agents include muteins and derivatives of the biologic agent, which may include, for example, fusion proteins, PEGylated derivatives, cholesterol-conjugated derivatives, etc., as known in the art. Also included are cytokine and cytokine receptor antagonists, e.g., traps and monoclonal antagonists, e.g., IL-1Ra, IL-1 trap, sIL-4Ra, etc. Also included are drugs that are biologically similar or bioequivalent to the active agents described herein.

[0230] Treatments for multiple sclerosis include corticosteroids, plasma exchange, ocrelizumab (Ocrevus®), IFN-β (Avonex®, Betaseron®, Rebif®, Extavia®, Plegridy®), glatiramer acetate (Copaxone®, Glatopa®), anti-VLA4 (Tysabri, natalizumab), dimethyl fumarate (Tecfidera®, Vumerity®), teriflunomide (Aubagio®), monomethyl fumarate (Bafiertam®), ozanimod (Zeposia®), and fluconazole (Valveolar). Drugs that may be used include fluticasone (fluticasone), fluoxetine (fluticasone), flucloxate (fluticasone), flucloxate (fluticasone), flucloxate (fluclox), flucloxone ...

[0231] The pharmaceutical composition administered to an individual contains an active agent, such as a therapeutic agent described above. The active ingredient is present in a therapeutically effective amount, i.e., an amount sufficient when administered to treat a disease or a condition mediated by it. The composition may also contain various other agents that enhance delivery and efficacy, e.g., enhance the delivery and stability of the active ingredient. Thus, for example, depending on the desired formulation, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances that approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, and detergents. The compositions may also include any of a variety of stabilizers, such as antioxidants.

[0232] The pharmaceutical compositions described herein can be administered in a variety of different ways, including by administering the composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, or intracranial means.

[0233] Such pharmaceutical compositions may be administered for the purpose of prevention (e.g., before diagnosis of a multiple sclerosis patient) or treatment (e.g., after diagnosis of a multiple sclerosis patient). Preventing, prophylaxis, or prevention of a disease or disorder, as used in the context of the present invention, refers to the administration of a composition that prevents the onset or development of multiple sclerosis or some or all of the symptoms of multiple sclerosis, or reduces the likelihood of the onset of the disease or disorder. Treating, treatment, or therapy of multiple sclerosis shall mean slowing, halting, or reversing disease progression by administering treatment according to the present invention. In a preferred embodiment, treating multiple sclerosis means reversing disease progression, ideally to the point of eliminating the disease itself.

[0234] VIII. Subject's Disease Activity The methods described herein focus on assessing a subject's disease activity by applying quantitative expression levels of biomarkers as input to a predictive model. In various embodiments, the subject is classified into a category based on the predicted assessment of disease activity. To classify the subject, the prediction for the subject may be compared with the results of individuals who have been previously classified into a clinically diagnosed category. For example, an individual may be clinically classified into one of the following: a diagnosis of MS (e.g., the presence of MS), a classification of MS subtype (e.g., radiologically isolated syndrome (RIS), clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary-progressive MS (SPMS)), a classification of quiescent or exacerbated status, a classification of disability level according to the Expanded Disability Status Scale (EDSS), a determined clinical response to treatment, and a clinical identification of risk for developing MS. Clinical categories may also be determined using the MS Functional Composite (MSFC), Timed 25-Foot Walk (T25Fw), 9-Hole Peg Test (9HPT), or patient-reported outcomes (e.g., Patient-Determined Disease Stage (PDDS) / MSSS (Patient-Derived Disability Status Scale), PRO Measurement Information System (PROMIS), or Multiple Sclerosis Rating Scale-Revised (MSRS-R)). Individuals may be clinically classified based on measurable measures of MS disease activity, such as the presence of a specific number of gadolinium-enhancing lesions (e.g., minimal disease activity) or at least one gadolinium-enhancing lesion (e.g., typical disease activity). Clinical classification may also be based on other radiological measures, including T2 lesions (new or enlarging), slowly enlarging lesions, rim-enlarging lesions, brain parenchymal fraction (BPF) and percent change, gray matter fraction, white matter fraction, thalamic volume, cortical gray matter volume, deep gray matter volume, or radiologist notes of ancillary features (e.g., Dawson's fingers). Previous classification of individuals may be based on clinical criteria.

[0235] Clinical diagnosis of MS may be made in a variety of ways. As an example, clinical diagnosis of MS may be made via magnetic resonance imaging (MRI) of the brain and spinal cord to identify lesions or plaques that form as a result of MS. The McDonald criteria may be used in making the diagnosis. Clinical diagnosis of MS may be made via lumbar puncture (spinal tap) to observe abnormalities in antibody concentrations in the cerebrospinal fluid due to the presence of MS. Clinical diagnosis of MS may also be made via evoked potential testing, in which electrical signals generated by neurons in the nervous system are recorded in response to a stimulus. Conduction disturbances indicate the presence of MS.

[0236] The clinical classification of a patient previously diagnosed with MS as being in a quiescent state versus a worsening state can depend on various factors. That is, a patient can be clinically classified as being in a worsening state after exhibiting a new disease associated with MS (e.g., a comorbidity or symptom such as clinical depression or optic neuritis). As another example, a patient can be clinically classified as being in a worsening state if they exhibit a significant worsening of symptoms. Examples include worsening balance and / or mobility, vision, eye pain, fatigue, and / or heart-related problems. A patient previously diagnosed with MS can be clinically classified as being in a quiescent state if they do not exhibit a new disease or a change or worsening of symptoms.

[0237] Determining whether a patient previously diagnosed with MS is responding to a therapy can depend on various clinical variables. For example, response to a therapy can be determined based on the occurrence or absence of relapses. If no relapse occurs, the patient can be considered to have responded to the therapy. Response to a therapy can also be determined based on the total number of relapses, the time to the first relapse, the patient's EDSS score, changes in the patient's EDSS score (e.g., an increase in score corresponds to no response to the therapy), or changes in MRI status (e.g., the occurrence of additional lesions or plaques corresponds to no response to the therapy).

[0238] Patients can be clinically classified into levels of disability, which can serve as a criterion for assessing disease progression. For example, the EDSS can be used to determine the severity of a patient's MS. Thus, patients are classified into categories corresponding to EDSS scores ranging from 1.0 to 10.0, in 0.5-point intervals. Typically, an EDSS score of 1.0 to 4.5 indicates an MS patient who is able to walk unaided. An EDSS score of 5.0 to 9.5 indicates an MS patient whose walking ability is impaired, with higher scores indicating greater disability. In various embodiments, an EDSS score of less than 6 indicates mild / moderate MS disease progression. In various embodiments, an EDSS score of 6 or greater indicates severe MS disease progression. In certain embodiments, an EDSS score of 0 to 3.0 indicates mild MS, an EDSS score of 3.5 to 5.5 indicates moderate MS, and an EDSS score of 6.0 to 9.5 indicates severe MS.

[0239] As another example, a patient may be clinically classified into levels of disability according to the PDDS, a scale validated as a self-report surrogate for the EDSS and therefore can be used to determine the severity of a patient's MS. A PDDS score of 0 indicates a normal level of disability with mild sensory symptoms and no activity limitations. A PDDS score of 1 indicates mild disability with only minor noticeable symptoms and minimal impact on lifestyle. A PDDS score of 2 indicates moderate disability, with no limitations on walking ability but significant problems that otherwise limit daily activities. A PDDS score of 3 indicates gait impairment that interferes with activities such as walking. A PDDS score of 4 indicates early cane disability, characterized by the use of a cane or one crutch for all or part of the walking period (e.g., being able to walk 25 feet in 20 seconds without a cane or crutch). A PDDS score of 5 indicates late cane disability, characterized by walking 25 feet using a cane or crutch. A PDDS score of 6 indicates bilateral disability, characterized by the need to use two canes, crutches, or a walker to walk 25 feet. A PDDS score of 7 indicates wheelchair / scooter disability, where the individual's primary means of transportation is a wheelchair / scooter. A PDDS score of 8 indicates bedridden disability, where the individual is unable to sit in a wheelchair for more than one hour. In various embodiments, a PDDS score of 4 or less indicates disease progression to mild / moderate MS disability. In various embodiments, a PDDS score greater than 4 indicates severe MS disease progression. In certain embodiments, a PDDS score of 0-1 indicates mild MS, a PDDS score of 2-4 indicates moderate MS, and a PDDS score of 5-8 indicates severe MS.

[0240] As another example, a patent may be clinically classified by level of disability according to the PROMIS assessment criteria. Generally, PROMIS scores are based on a T-score metric, with a score of 50 representing the mean score of a corresponding reference population with a standard deviation of 10. Thus, an individual's score of 40 indicates that the individual is one standard deviation below the mean of the corresponding reference population (e.g., a score of 40 indicates that the individual's MS disability is one standard deviation below the population mean MS disability). An individual's score of 60 indicates that the individual is one standard deviation above the mean of the corresponding reference population (e.g., a score of 60 indicates that the individual's MS disability is one standard deviation above the population mean MS disability).

[0241] As another example, patents may be clinically classified by level of impairment according to the MSRS-R scale. Generally, MSRS-R scores may be measured according to the following items: 1) walking; 2) arm and hand use; 3) vision (if using glasses or contacts); 4) speaking clearly; 5) swallowing; 6) thinking, memory, or cognition; 7) numbness, tingling, burning, or pain; and 8) bowel or bladder. Each item is rated according to the level of impairment: 0—normal; 1—symptoms with no impairment; 2—mild impairment requiring no assistance; 3—moderate impairment requiring assistance; and 4—complete loss of function, requiring maximum assistance. The total MSRS-R score represents the sum of the scores across the eight items.

[0242] IX. Computer Implementation Methods of the invention, including methods of assessing multiple sclerosis activity (eg, multiple sclerosis disease progression) in an individual, are, in some embodiments, implemented on one or more computers.

[0243] For example, the construction and deployment of the predictive model and database storage can be implemented in hardware or software, or a combination of both. In one embodiment of the present invention, a machine-readable storage medium is provided, the medium including data storage material encoded with machine-readable data, which, when used with a machine programmed with instructions for using the data, can display any of the datasets and the execution and results of the predictive model of the present invention. Such data can be used for various purposes, such as patient monitoring, treatment considerations, etc. The present invention can be implemented in a computer program executed on a programmable computer including a processor, a data storage system (including volatile and non-volatile memory and / or storage elements), a graphics adapter, a pointing device, a network adapter, at least one input device, and at least one output device. A display is connected to the graphics adapter. The program code is applied to the input data to perform the functions described above and generate output information. The output information is applied to one or more output devices in a known manner. The computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.

[0244] Each program can be implemented in a high-level procedural or object-oriented programming language to communicate with a computer system. However, if desired, the program can be implemented in assembly or machine language. In either case, the language can be a compiled or interpreted language. Each such computer program for configuring and operating a computer is preferably stored on a general-purpose or special-purpose programmable computer-readable storage medium or device (e.g., ROM or magnetic diskette) when the storage medium or device is read by a computer that performs the procedures described herein. It is also contemplated that the system can be implemented as a computer-readable storage medium configured with a computer program that causes the computer to operate in a specific, predefined manner to perform the functions described herein.

[0245] The signature patterns and their databases can be provided in various media to facilitate their use. "Media" refers to a product containing the signature pattern information of the present invention. The database of the present invention can be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROM; and hybrids of these fields, such as magnetic / optical storage media. Those skilled in the art will readily understand how any existing computer-readable medium can be used to create a product containing the storage of the database information. "Recorded" refers to the process of storing information on a computer-readable medium using methods known in the art. Any convenient data storage structure can be selected based on the means used to access the stored information. For example, various data processor programs and formats, such as word processing text files and database formats, can be used for storage.

[0246] In some embodiments, the methods of the present invention, including methods for assessing multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) in an individual, are implemented on one or more computers in a distributed computing system environment (e.g., a cloud computing environment). For purposes of this description, "cloud computing" is defined as a model for enabling on-demand network access to a shared set of configurable computing resources. Cloud computing can be used to provide on-demand access to a shared set of configurable computing resources. The shared set of configurable computing resources can be rapidly provisioned via virtualization, released with little administrative effort or service provider intervention, and scaled accordingly. Cloud computing models can comprise various features, such as on-demand self-service, wide-area network access, resource sharing, rapid elasticity, and service metering. Cloud computing models can also expose various service models, such as software as a service ("SaaS"), platform as a service ("PaaS"), and infrastructure as a service ("IaaS"). Cloud computing models can also be deployed using various deployment models, such as private clouds, community clouds, public clouds, and hybrid clouds. In this description and claims, a "cloud computing environment" is an environment in which cloud computing is used.

[0247] VIII.A. Exemplary Computer 8 illustrates an exemplary computer 800 for implementing the entities shown in FIGS. 1 and 3. Computer 800 includes at least one processor 802 connected to a chipset 804. Chipset 804 includes a memory controller hub 820 and an input / output (I / O) controller hub 822. Memory 806 and a graphics adapter 812 are connected to memory controller hub 820, and a display 818 is connected to graphics adapter 812. Storage devices 808, input devices 814, and a network adapter 816 are connected to I / O controller hub 822. Other embodiments of computer 800 have different architectures.

[0248] The storage device 808 is a non-transitory computer-readable storage medium, such as a hard drive, a compact disc read-only memory (CD-ROM), a DVD, or a solid-state memory device. The memory 806 holds instructions and data used by the processor 802. The input interface 814 is a touchscreen interface, a mouse, a trackball, or other type of pointing device, a keyboard, or some combination thereof, and is used to input data into the computer 800. In some embodiments, the computer 800 may be configured to receive input (e.g., commands) from the input interface 814 via gestures from a user. The graphics adapter 812 displays images and other information on the display 818. The network adapter 816 connects the computer 800 to one or more computer networks.

[0249] The computer 800 is configured to execute computer program modules to provide the functionality described herein. As used herein, the term "module" refers to computer program logic used to provide a particular function. Thus, a module may be implemented in hardware, firmware, and / or software. In one embodiment, the program modules are stored in the storage device 808, loaded into the memory 806, and executed by the processor 802.

[0250] 1 may vary depending on the embodiment and the processing power required by the entities. For example, disease progression system 130 may run on a single computer 800 or multiple computers 800 that communicate with each other over a network, such as a server farm. Computer 800 may lack some of the components described above, such as graphics adapter 812 and display 818.

[0251] X. Kit Implementation Also disclosed herein are kits for assessing multiple sclerosis disease activity (e.g., multiple sclerosis disease progression) in an individual. Such kits may include reagents for detecting expression levels of one or more biomarkers and instructions for assessing disease activity (e.g., multiple sclerosis disease progression) based on the detected expression levels.

[0252] The detection reagents may be provided as part of a kit. Accordingly, the present invention further provides kits for detecting the presence of a panel of biomarkers of interest in a biological test sample. The kit may include a set of reagents for generating a dataset via at least one protein detection assay (e.g., an immunoassay) analyzing a test sample from a subject. In various embodiments, the set of reagents allows for the detection of quantitative expression levels of biomarkers from any one of Tables 4-6. In certain embodiments, the set of reagents allows for the detection of quantitative expression levels of biomarkers classified as Tier A, Tier B, or Tier C biomarkers in Table 1. In certain embodiments, the set of reagents allows for the detection of quantitative expression levels of biomarkers classified as Tier 1, Tier 2, or Tier 3 biomarkers in Table 2. In certain aspects, the set of reagents allows for the detection of quantitative expression levels of biomarkers classified as Tier 1, Tier 2, or Tier 3 biomarkers in Table 3. In certain aspects, the reagents include one or more antibodies that bind to one or more of the markers. The antibodies may be monoclonal or polyclonal. In some embodiments, the reagents may include reagents for performing an ELISA, including a buffer and a detection agent.

[0253] The kit can include instructions for use of the set of reagents. For example, the kit can include instructions for performing at least one biomarker detection assay, such as an immunoassay, a protein-binding assay, an antibody-based assay, an antigen-binding protein-based assay, a protein-based array, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein array, a blot, a Western blot, nephelometry, turbidimetry, chromatography, mass spectrometry, an enzyme activity, a proximity extension assay, and an immunoassay selected from RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassay, and immunoprecipitation.

[0254] In various embodiments, the kit includes instructions for practicing the methods disclosed herein (e.g., methods for training or deploying a predictive model to predict an assessment of disease activity, such as multiple sclerosis disease progression). These instructions may be present in the kit in a variety of formats, one or more of which may be present in the kit. One form in which these instructions may be present is printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, in the kit's packaging, in a package insert, etc. Yet another means would be a computer-readable medium on which the information is recorded, such as a diskette, CD, hard drive, network data storage, etc. Yet another means that may be present is a website address that can be used via the Internet to access the information on the removed site. Any convenient means may be present in the kit.

[0255] XI. System Further disclosed herein are systems for analyzing quantitative expression levels of biomarkers to assess disease activity (e.g., multiple sclerosis disease progression). In various embodiments, such systems may comprise a set of reagents for detecting expression levels of biomarkers in a biomarker panel, an apparatus configured to receive a mixture of the set of reagents and a test sample obtained from a subject to measure the expression level of a soluble mediator, and a computer system communicatively coupled to the apparatus for obtaining the measured expression levels and for implementing a predictive model to assess disease activity (e.g., multiple sclerosis disease progression).

[0256] The set of reagents allows for the detection of quantitative expression levels of biomarkers in a biomarker panel. In various embodiments, the set of reagents includes reagents used to perform an assay, such as an assay or immunoassay, as described above. For example, the reagents include one or more antibodies that bind to one or more of the biomarkers. The antibodies may be monoclonal or polyclonal. As another example, the reagents may include reagents for performing an ELISA, including a buffer and a detection agent.

[0257] The device is configured to detect the expression level of a biomarker in a mixture of reagents and a test sample. For example, the device may determine the quantitative expression level of a biomarker through an immunological assay or an assay for nucleic acid detection. The mixture of reagents and a test sample may be provided to the device via various conduits, examples of which include wells in a well plate (e.g., a 96-well plate), vials, tubes, and integrated fluidic circuits. Thus, the device may have an opening (e.g., a slot, cavity, opening, slide tray) that can receive a container containing the reagent-test sample mixture and perform a reading to generate a quantitative expression value of the biomarker. Examples of devices include plate readers (e.g., luminescence plate readers, absorbance plate readers, fluorescence plate readers), spectrometers, and spectrophotometers.

[0258] A computer system, such as the exemplary computer 800 depicted in Figure 8, is in communication with the device to receive the quantitative expression values ​​of the biomarkers. The computer system implements a predictive model that analyzes the quantitative expression values ​​of the biomarkers in silico to predict an assessment of disease activity (e.g., multiple sclerosis disease progression).

[0259] XII. Additional Embodiments Further disclosed herein is a method for predicting multiple sclerosis progression in a subject, the method comprising obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises each biomarker of at least one group selected from Group 1, Group 2, and Group 3, wherein Group 1 comprises biomarker 1, biomarker 2, biomarker 3, biomarker 4, biomarker 5, biomarker 6, biomarker 7, and biomarker 8, and wherein biomarker 1 is selected from the group consisting of NEFL, NEFL, NEFL+ ... , MOG, CADM3, or GFAP; biomarker 1 is MOG, CADM3, KLK6, BCAN, OMG, or GFAP; biomarker 3 is CD6, CD5, CRTAM, CD244, or TNFRSF9; biomarker 4 is CXCL9, CXCL10, IL-12B, CXCL11, or GFAP; biomarker 5 is OPG, TFF3, or ENPP2; biomarker 6 is OPN, OMD, MEPE, or GFAP; and biomarker 7 is CXCL13, NOS3, or M MP-2; biomarker 8 is GFAP, NEFL, OPN, CXCL9, MOG, or CHI3L1; group 2 includes biomarker 9, biomarker 10, biomarker 11, biomarker 12, biomarker 13, biomarker 14, biomarker 15, and biomarker 16; biomarker 9 is CDCP1, IL-18BP, IL-18, GFAP, or MSR1; biomarker 10 is CCL20, CCL3, or TWEAK; and biomarker 11 is IL-12B, IL12A, or CXCL9; biomarker 12 is APLP1, SEZ6L, BCAN, DPP6, NCAN, or KLK6; biomarker 13 is TNFRSF10A, TNFRSF11A, SPON2, CHI3L1, or IFI30; biomarker 14 is SERPINA9, TNFRSF9, or CNTN4; biomarker 15 is FLRT2, DDR1, NTRK2, CDH6, MMP-2; and biomarker 16 is TNFSF13B, CXCL16, ALCAM, or IL-18; and Group 3 isbiomarker 17 is GH, GH2, or IGFBP-1; biomarker 18 is VCAN, TINAGL1, CANT1, NECTIN2, MMP-9, or NPDC1; biomarker 19 is PRTG, NTRK2, NTRK3, or CNTN4; and biomarker 20 is CNTN2, DPP6, GDNFR-α3, or SCARF2; and generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers.

[0260] In various embodiments, the plurality of biomarkers includes each biomarker in Group 1, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 3 is CD6, biomarker 4 is CXCL9, biomarker 5 is OPG, biomarker 6 is OPN, biomarker 7 is CXCL13, and biomarker 8 is GFAP. In various embodiments, the plurality of biomarkers further includes each biomarker in Group 2, where biomarker 9 is CDCP1, biomarker 10 is CCL20, biomarker 11 is IL-12B, biomarker 12 is APLP1, biomarker 13 is TNFRSF10A, biomarker 14 is SERPINA9, biomarker 15 is FLRT2, and biomarker 16 is TNFSF13B. In various embodiments, the plurality of biomarkers further includes each biomarker in Group 3, wherein biomarker 17 is GH, biomarker 18 is VCAN, biomarker 19 is PRTG, and biomarker 20 is CNTN2.

[0261] In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R 2 In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value.

[0262] In various embodiments, the plurality of biomarkers comprises biomarkers from Group 1 comprising biomarker 2 and biomarker 8, where biomarker 2 is MOG and biomarker 8 is GFAP. In various embodiments, the plurality of biomarkers comprises biomarkers from Group 1 comprising biomarker 1 and biomarker 8, where biomarker 1 is NEFL and biomarker 8 is GFAP. In various embodiments, the plurality of biomarkers comprises biomarkers from Group 1 comprising biomarker 8 and biomarker 2 comprising biomarker 12, where biomarker 8 is GFAP and biomarker 12 is APLP1. In various embodiments, the plurality of biomarkers comprises biomarkers from Group 1 comprising biomarker 1 and biomarker 2, where biomarker 1 is NEFL and biomarker 2 is MOG. In various embodiments, the plurality of biomarkers comprises biomarkers from Group 1 comprising biomarker 1, biomarker 2, and biomarker 8, where biomarker 1 is NEFL, biomarker 2 is MOG, and biomarker 8 is GFAP. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, which include biomarker 1 and biomarker 2, and Group 3 biomarkers, which include biomarker 18, where biomarker 1 is NEFL, biomarker 2 is MOG, and biomarker 18 is GH. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, which include biomarker 1 and biomarker 2, and Group 2 biomarkers, which include biomarker 15, where biomarker 1 is NEFL, biomarker 2 is MOG, and biomarker 15 is SERPINA9. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, which include biomarker 6 and biomarker 7, and Group 2 biomarkers, which include biomarker 15, where biomarker 6 is OPN, biomarker 7 is CXCL13, and biomarker 15 is SERPINA9.In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers comprising biomarkers 6 and biomarker 7, and Group 2 biomarkers comprising biomarker 13, where biomarker 6 is OPN, biomarker 7 is CXCL13, and biomarker 13 is TNFRSF10A. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers comprising biomarkers 1, biomarker 2, and biomarker 8, and Group 2 biomarkers comprising biomarker 11, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 8 is GFAP, and biomarker 11 is IL-12B. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers comprising biomarkers 1, biomarker 2, and biomarker 8, and Group 3 biomarkers comprising biomarker 20, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 8 is GFAP, and biomarker 20 is PRTG. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, which includes biomarker 1, biomarker 2, and biomarker 8, and Group 2 biomarkers, which includes biomarker 12, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 8 is GFAP, and biomarker 12 is APLP1. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, which includes biomarker 1 and biomarker 2, Group 2 biomarkers, which includes biomarker 15, and Group 3 biomarkers, which includes biomarker 18, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 15 is SERPINA9, and biomarker 18 is GH.In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, including biomarker 1 and biomarker 2, and Group 2 biomarkers, including biomarker 13 and biomarker 15, where biomarker 1 is NEFL, biomarker 2 is MOG, biomarker 13 is TNFRSF10A, and biomarker 15 is SERPINA9. In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers, including biomarker 3, and Group 2 biomarkers, including biomarker 10, biomarker 11, and biomarker 12, where biomarker 3 is CD6, biomarker 10 is CCL20, biomarker 11 is IL-12B, and biomarker 12 is APLP1. In various embodiments, the prediction of multiple sclerosis disease progression is the Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6 or greater indicates severe MS disease progression.

[0263] In various embodiments, the plurality of biomarkers comprises biomarkers of Group 1 comprising biomarker 1, biomarker 3, and biomarker 7, where biomarker 1 is NEFL, biomarker 3 is CD6, and biomarker 7 is CXCL13. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) of at least 0.8. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) of at least 0.9. In various embodiments, the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

[0264] In various embodiments, the plurality of biomarkers comprises Group 2 biomarkers comprising biomarker 9 and biomarker 11, where biomarker 9 is CDCP1 and biomarker 11 is IL-12B. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

[0265] In various embodiments, the plurality of biomarkers comprises Group 1 biomarkers including biomarker 3, Group 2 biomarkers including biomarker 9 and biomarker 11, and Group 3 biomarkers including biomarker 19, where biomarker 3 is CD6, biomarker 9 is CDCP1, biomarker 11 is IL-12B, and biomarker 19 is VCAN. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0266] Further disclosed herein is a method for predicting multiple sclerosis progression in a subject, the method comprising: obtaining, or having obtained, a dataset comprising expression levels of a plurality of biomarkers, including one or more neurodegenerative biomarkers selected from the group consisting of NEFL, APLP1, OPG, SERPINA9, PRTG, GFAP, CNTN2, and FLRT2; one or more inflammatory biomarkers selected from the group consisting of CCL20, GH, CXCL13, IL-12B, VCAN, TNFRSF10A, TNFSF13B, CD6, and CXCL9; one or more immunomodulatory biomarkers selected from the group consisting of CDCP1 and OPN; or one or more myelin integrity biomarkers selected from the group consisting of MOG; and applying a predictive model to the expression levels of the plurality of biomarkers to generate a prediction of multiple sclerosis progression.

[0267] In various embodiments, the one or more neurodegeneration biomarkers include NEFL, OPG, and GFAP, the one or more inflammation biomarkers include CXCL13, CD6, and CXCL9, the one or more immunomodulatory biomarkers include OPN, and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers further include APLP1, SERPINA9, and FLRT2, the one or more inflammation biomarkers further include CCL20, IL-12B, TNFRSF10A, and TNFSF13B, and the one or more immunomodulatory biomarkers further include CDCP1. In various embodiments, the one or more neurodegeneration biomarkers further include PRTG and CNTN2, and the one or more inflammation biomarkers include GH and VCAN. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.517. 2 In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value.

[0268] In various embodiments, the one or more myelin integrity biomarkers include MOG and the one or more neurodegeneration biomarkers include GFAP. In various embodiments, the one or more neurodegeneration biomarkers include NEFL and GFAP. In various embodiments, the one or more neurodegeneration biomarkers include GFAP and APLP1. In various embodiments, the one or more neurodegeneration biomarkers include NEFL and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers include NEFL and GFAP and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers include NEFL, the one or more myelin integrity biomarkers include MOG, and the one or more inflammation biomarkers include GH. In various embodiments, the one or more neurodegeneration biomarkers include NEFL and SERPINA9 and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more immune-modulating biomarkers include OPN, the one or more inflammation biomarkers include CXCL13, and the one or more neurodegeneration biomarkers include SERPINA9. In various embodiments, the one or more immune-modulating biomarkers include OPN, and the one or more inflammation biomarkers include CXCL13 and TNFRSF10A. In various embodiments, the one or more neurodegeneration biomarkers include NEFL, the one or more myelin integrity biomarkers include MOG, and the one or more inflammatory biomarkers include IL-12B. In various embodiments, the one or more neurodegeneration biomarkers include NEFL, PRTG, and GFAP, and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers include NEFL, APLP1, and GFAP, and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers include NEFL and SERPINA9, the one or more inflammation biomarkers include GH, and the one or more myelin integrity biomarkers include MOG.In various embodiments, the one or more neurodegeneration biomarkers include NEFL and SERPINA9, the one or more inflammation biomarkers include TNFRSF10A, and the one or more myelin integrity biomarkers include MOG. In various embodiments, the one or more neurodegeneration biomarkers include APLP1, and the one or more inflammation biomarkers include CCL20, IL-12B, and CD6. In various embodiments, the prediction of multiple sclerosis disease progression is the Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6.0 or greater indicates severe MS disease progression.

[0269] In various embodiments, the one or more neurodegenerative biomarkers include NEFL and the one or more inflammatory biomarkers include CD6 and CXCL13. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) value of at least 0.8. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) of at least 0.9. In various embodiments, the prediction of multiple sclerosis disease progression is a patient-determined disease stage (PDDS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a patient-determined disease stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a patient-determined disease stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

[0270] In various embodiments, the one or more inflammatory biomarkers include IL-12B and the one or more immunomodulatory biomarkers include CDCP1. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

[0271] In various embodiments, the one or more inflammatory biomarkers include CD6, IL-12B, and VCAN, and the one or more immunomodulatory biomarkers include CDCP1. In various embodiments, the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0272] Further disclosed herein is a method for predicting multiple sclerosis progression in a subject, the method comprising obtaining, or having obtained, a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises two or more of NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20, IL-12B, APLP1, TNFRSFlOA, SERPINA9, FLRT2, TNFSF13B, GH, VCAN, PRTG, and CNTN2; and generating a prediction of multiple sclerosis disease activity by applying a predictive model to the expression levels of the plurality of biomarkers. In various embodiments, the plurality of biomarkers comprises each of NEFL, MOG, CD6, CXCL9, OPG, OPN, CXCL13, GFAP, CDCP1, CCL20, IL-12B, APLP1, TNFRSFlOA, SERPINA9, FLRT2, TNFSF13B, GH, VCAN, PRTG, and CNTN2. In various embodiments, the performance of the predictive model is evaluated by a correlation coefficient (R) of at least 0.517. 2 In various embodiments, the predictor of multiple sclerosis disease progression is a brain parenchymal fraction value.

[0273] In various embodiments, the plurality of biomarkers includes MOG and GFAP. In various embodiments, the plurality of biomarkers includes NEFL and GFAP. In various embodiments, the plurality of biomarkers includes GFAP and APLP1. In various embodiments, the plurality of biomarkers includes NEFL and MOG. In various embodiments, the plurality of biomarkers includes NEFL, MOG, and GFAP. In various embodiments, the plurality of biomarkers includes NEFL, MOG, and GH. In various embodiments, the plurality of biomarkers includes NEFL, MOG, and SERPINA9. In various embodiments, the plurality of biomarkers includes OPN, CXCL13, and SERPINA9. In various embodiments, the plurality of biomarkers includes OPN, CXCL13, and TNFRSF10A. In various embodiments, the plurality of biomarkers includes NEFL, MOG, GFAP, and IL-12B. In various embodiments, the plurality of biomarkers includes NEFL, MOG, GFAP, and PRTG. In various embodiments, the plurality of biomarkers includes NEFL, MOG, GFAP, and APLP1. In various embodiments, the plurality of biomarkers comprises NEFL, MOG, SERPINA9, and GH. In various embodiments, the plurality of biomarkers comprises NEFL, MOG, TNFRSF10A, and SERPINA9. In various embodiments, the plurality of biomarkers comprises CD6, CCL20, IL-12B, and APLP1. In various embodiments, the prediction of multiple sclerosis disease progression is the Expanded Disability Status Scale (EDSS) score. In various embodiments, an Expanded Disability Status Scale (EDSS) score of 6 or less indicates mild / moderate MS disease progression, and an EDSS score of greater than 6.5 indicates severe MS disease progression.

[0274] In various embodiments, the plurality of biomarkers comprises NEFL, CD6, and CXCL13. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) value of at least 0.8. In various embodiments, the performance of the predictive model is characterized by an area under the ROC curve (AUROC) of at least 0.9. In various embodiments, the prediction of multiple sclerosis disease progression is a patient-determined disease staging (PDDS) score. In various embodiments, the prediction of multiple sclerosis disease progression is a patient-determined disease staging (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression. In various embodiments, a patient-determined disease staging (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

[0275] In various embodiments, the plurality of biomarkers comprises CDCP1 and IL-12B. In various embodiments, the predictor of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

[0276] In various embodiments, the plurality of biomarkers comprises CD6, CDCP1, IL-12B, and VCAN. In various embodiments, the predictor of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

[0277] In various embodiments, generating a prediction of multiple sclerosis disease progression by applying the predictive model to expression levels of the plurality of biomarkers further includes applying the predictive model to one or more subject attributes of the subject, where the subject attributes include any of age, sex, and disease duration. In various embodiments, generating a prediction of multiple sclerosis disease progression includes comparing the score output by the predictive model to a reference score. In various embodiments, the reference score corresponds to any of the following: A) EDSS score; B) brain parenchymal fraction value; C) PDDS score; D) PROMIS score; or E) MSRS-R score. In various embodiments, the reference score further corresponds to mild / moderate MS disease progression or severe MS disease progression.

[0278] In various embodiments, the expression levels of the plurality of biomarkers are determined from a test sample obtained from the subject. In various embodiments, the test sample is a blood or serum sample. In various embodiments, the subject has, is suspected of having, or has previously been diagnosed with multiple sclerosis.

[0279] In various embodiments, obtaining or obtaining the dataset comprises performing an immunoassay to determine expression levels of a plurality of biomarkers. In various embodiments, the immunoassay is a proximity extension assay (PEA) or a LUMINEX xMAP multiplex assay. In various embodiments, performing the immunoassay comprises contacting the test sample with a plurality of reagents including antibodies. In various embodiments, the antibodies comprise one of monoclonal and polyclonal antibodies. In various embodiments, the antibodies comprise both monoclonal and polyclonal antibodies. In various embodiments, the method further comprises selecting a therapy to administer to the subject based on the prediction of multiple sclerosis disease progression. In various embodiments, the method further comprises determining the therapeutic efficacy of a therapy previously administered to the subject based on the prediction of multiple sclerosis disease progression. In various embodiments, determining the therapeutic efficacy of a therapy comprises comparing the prediction to a previous prediction determined for the subject at a previous time point. In various embodiments, determining the therapeutic efficacy of a therapy comprises determining that the therapy is effective depending on the difference between the prediction and the previous prediction. In various embodiments, determining the therapeutic efficacy of a therapy comprises determining that the therapy lacks efficacy in response to a lack of difference between the prediction and said previous prediction. [Example]

[0280] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for.

[0281] Example 1: Human Clinical Study Multiple human clinical studies were used to develop this biomarker panel, including: ACP = Accelerated Care Project (study code: F2), CLIMB = Comprehensive Longitudinal Investigation of Multiple Sclerosis at Brigham and Women's Hospital (study codes: F3A, F3B, F3C, and F4), EPIC = Expression, Proteomics, Imaging, and Clinical at UCSF (study code: F5), and UHBC = University Hospital of Basel Cohort (study code: F6). ACP (n = 124) focused on clinically defined exacerbation events, while AIM1 (n = 60) and F4 (unpaired) (n = 326) focused on annualized relapse rates (ARR). AIM3 (unpaired) (n = 58), F4 (unpaired) (n = 326), and F5 EPIC (n = 180) focus on a cross-sectional view of gadolinium (Gd)-enhanced MRI lesion endpoints, while AIM3 (paired) (n = 58), F4 (paired) (n = 196), and F6 (n = 205) do the same through longitudinal analysis (e.g., considering patient pairs to establish baseline normals). Additionally, in some scenarios, biomarker analysis was performed by combining samples from various studies with the same endpoint. For example, study code F4 and study code F5, which share a common endpoint—the presence or absence of gadolinium-enhancing lesions—were combined for biomarker analysis. Within each of the three broad categories, each sample was equally weighted across the seven study paradigms (i.e., studies with more samples received proportionally more weight). Study codes and other information for the analysis are documented in Tables 5A and 5B below. Overall, two immunoassay platforms (rules-based medicine (RBM) and Olink biomarker panels) were used to screen over 1300 proteins across over 1000 individual samples. Multiple endpoints were investigated, including the presence / absence of gadolinium-enhancing lesions, clinical recurrence status, EDSS, annual recurrence rate, and T2 volume.

[0282] Example 2: Univariate analysis For the univariate analysis of individual biomarkers, three different statistical evaluation criteria were calculated. 1) Univariate populations - p-values ​​for standard statistical tests The p-values ​​were converted to t-statistics using the conventional inverse norm function, and then the statistics were combined based on the respective sample sizes using Stauffer's method. The final p-value / test statistic, which reflects cumulative power, was then normalized to a [0,1] scale.

[0283] 2) AUC value of the integral of the true positive rate and false positive rate of the univariate separation-ROC curve AUCs were calculated for each individual marker in the selected dataset, then normalized to a [0,1] scale, and their weighted sum (based on sample size below) was then used to convert the cumulative separation power into a single value between 0 and 1.

[0284] 3) Univariate regression - Adjusted R-squared value of OLS between lesion burden (removing the top lesion count (e.g., 5 lesions) to exclude outliers) and distribution of normalized protein expression values This was done for each cohort independently as well as for the mixed cohort in the training / test split.

[0285] Example 3: Multivariate analysis For multivariate biomarker analysis, the following was performed. 1) Multivariate biomarker ranking - We combined accuracy-weighted aggregate importance across millions of simulated support vector machines, logistic regression, random forests, linear discriminant analysis, and stochastic gradient descent models with various feature sizes. Grid search exhausted hyperparameter tuning (e.g., regularization or choice of numerical solver method). Forward selection: We simulated building multivariate models thousands of times and then summed the most commonly selected biomarkers across various cross-validated slices of the dataset. We iteratively incorporated forward feature selection using optimization metrics (e.g., AUC, Fl for classification, and adjusted R2, RMSE for regression). Because multiple data cohorts were involved, the importance of each study was weighted by sample size, and Gd was ranked as the primary endpoint. A spatially constrained 21-plex was then completed. Next, specific features were sequentially eliminated, and after feature elimination, the remaining features still had predictive power for each endpoint / study. Cross-validation and bootstrapping were used to reduce overfitting. When possible, a test holdout set was used. 2) Bivariate (two-feature) models to explore the improvement of orthogonal signals 3) Interaction terms (products, ratios, quadratic terms)

[0286] To ensure repeatable results: The model was trained on the CLIMB dataset and tested on the EPIC dataset (before and after batch normalization and demographic adjustment). Paired samples between AIM3 and F4 (baseline normalized signals)

[0287] Example 4: Univariate APLP1 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of APLP1 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10A below.

[0288] Example 5: Univariate CCL20 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CCL20 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10B below.

[0289] Example 6: Univariate CD6 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CD6 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10C below.

[0290] Example 7: Univariate CDCP1 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CDCP1 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10D below.

[0291] Example 8: Univariate CNTN2 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CNTN2 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10E below.

[0292] Example 9: Univariate COL4A1 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of COL4A1 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10F below.

[0293] Example 10: Univariate CXCL9 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CXCL9 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10G below.

[0294] Example 11: Univariate FLRT2 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CDCP1 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10H below.

[0295] Example 12: Univariate growth hormone biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of growth hormone (GH) was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10I below.

[0296] Example 13: Univariate IFI30 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of IFI30 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10J below.

[0297] Example 14: Univariate MOG biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of MOG was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10K below.

[0298] Example 15: Univariate NEFL biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of NEFL was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10L below.

[0299] Example 16: Univariate OPG biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of OPG was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10M below.

[0300] Example 17: Univariate OPN biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of OPN was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10N below.

[0301] Example 18: Univariate PRTG biomarker analysis for predicting multiple sclerosis disease activity Univariate analyses of PRTG were performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analyses (p-values, area under the curve (AUC), and correlation values ​​(R-squared)) are shown in Table 10O below.

[0302] Example 19: Univariate SERPINA9 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of SERPINA9 was performed in various human clinical studies according to the methods described in Example 2. Statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown below in Table 10P.

[0303] Example 20: Univariate TNFRSF10A biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of TNFRSF10A was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10Q below.

[0304] Example 21: Univariate VCAN biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of VCAN was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10R below.

[0305] Example 22: Univariate CHI3L1 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CHI3L1 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10S below.

[0306] Example 23: Univariate CXCL13 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of CXCL13 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10T below.

[0307] Example 24: Univariate growth hormone 2 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of growth hormone 2 (GH2) was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10U below.

[0308] Example 25: Univariate IL-12B biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of IL-12B was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10V below.

[0309] Example 26: Univariate IL18 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of IL18 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10W below.

[0310] Example 27: Univariate MMP-2 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of MMP-2 was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10X below.

[0311] Example 28: Univariate MMP-9 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of MMP-9 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10Y below.

[0312] Example 29: Univariate VCAM-1 biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of VCAM-1 was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10Z below.

[0313] Example 30: Univariate TNFSF13B biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of TNFSF13B was performed in various human clinical studies according to the method described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10AA below.

[0314] Example 31: Univariate GFAP biomarker analysis for predicting multiple sclerosis disease activity Univariate analysis of GFAP was performed in various human clinical studies according to the methods described in Example 2. The statistical evaluation criteria of the univariate analysis (p-value, area under the curve (AUC), and correlation value (R-squared)) are shown in Table 10BB below.

[0315] Example 32: Univariate biomarker analysis for predicting different types of multiple sclerosis disease activity Univariate analysis of various biomarkers across various human clinical studies was performed according to the methods described in Example 2. Statistical p-values ​​and R 2 Values ​​are shown in Table 11 below for each of the individual biomarkers associated with various MS disease activity endpoints.

[0316] Example 33: Model training and validation - baseline normalization shift A linear regression model (L2 ridge regularization) was trained to predict the baseline normalized shift of Gd analysis in two different populations (F4 and F6), where the model was trained to classify sample pairs with and without Gd-enhancing MRI lesions appearing compared to baseline (both sera were collected close to the MRI).

[0317] A logistic regression classification algorithm was applied to the positive vs. negative shift in Gd counts. Because the predictor is the shift in biomarker protein levels (from a Gd-inactive baseline sample on the corresponding MRI), demographic adjustment is not necessary because intrapatient variability is accounted for. The dataset was divided into training and 5-fold cross-validation sets, and model parameters were hypertuned to optimize the model; all biomarkers in the custom assay panel (Tiers 1, 2, and 3 in Table 2 below) were included as model features. The area under the curve (AUROC) of the receiver operating characteristic obtained from the 5-fold cross-validation for classifying Gd shifts is 0.958 + / - 0.034.

[0318] Figures 2A and 2B show the sequential progressive selection performance profiles of the F6 and F4 studies, respectively.

[0319] Next, a separate training / test holdout of the four-feature model was trained and tested. Here, a logistic regression (L1 regularized) model was trained on the F6 data and tested on the F4 data. The model's feature space was restricted to the top four features in the analysis. A logistic regression classification algorithm was applied to the positive vs. negative shift in Gd counts. Because the predictor is the shift in biomarker protein levels (from a Gd-inactive baseline), demographic correction is not necessary because within-patient variability is accounted for. To utilize the same model coefficients for both the training and test sets, bridge normalization was applied to the dataset using a duplicate set of re-run samples. To optimize the model, the model parameters were hypertuned; all biomarkers in the custom assay panel were included as model features. This analysis was performed to evaluate multivariate prediction performance (compared to the univariate performance of serum neurofilament light chain, or sNfL), and is shown in Figure 3A. The model demonstrated an AUROC of 0.91 (sNfL -0.88), accuracy of 0.84 (sNfL -0.77), sensitivity of 0.76 (sNfL -0.70), specificity of 0.91 (sNfL -0.83), and Youden's statistic of 0.67 (sNfL -0.53). Figure 3B shows the corresponding confusion matrix of the multivariate classifier, which establishes a high true positive rate (TPR) of 0.757 and a high true negative rate (TNR) of 0.905.

[0320] Example 34: Model training and validation - cross-sectional classification of disease presence / absence A logistic regression model (L1 regularization) was trained to predict the presence or absence of disease based on Gd counts. Specifically, the model was trained to predict minimal disease (corresponding to one or no lesions per MRI), typical disease (corresponding to one or no lesions), and extreme disease (corresponding to three or more lesions or no lesions). 321 samples from F4, 180 samples from F5, and 155 samples from F6 were used to build the model. Figure 4A shows the sequential forward selection performance profiles.

[0321] To build the models, bridge normalization and demographic correction were applied to the dataset resulting from the merge of the three studies. The dataset was divided into training and 5-fold cross-validation sets, and the model parameters were hypertuned to optimize the model; all biomarkers in the custom assay panel were included as model features. Figure 4B shows the receiver operating characteristic curves (ROC) curves for the three models. Specifically, the AUROCs resulting from the 5-fold cross-validation were 0.741 ± 0.017 for the minimal model, 0.785 ± 0.004 for the normal model, and 0.903 ± 0.009 for the extreme model. Further gadolinium-based classification predictions are shown in Table 12.

[0322] Additionally, Figure 4C shows the normalized confusion matrix for each model. A comparison of Gd+ classification is shown below using 1) the univariate NFL model, 2) the multivariate model including NFL, and 3) the multivariate model excluding NFL.

[0323] Example 35: Model training and validation - Predicting disease severity by predicting lesion counts A linear regression model (L2 regularization) was trained and tested using 321 samples from F4, 180 samples from F5, and 155 samples from F6. A ridge regression algorithm was applied to the Gd counts. Bridge normalization and demographic correction were applied to the dataset resulting from the merge of the three studies. The dataset was split into training and 5-fold cross-validation sets; all biomarkers from the custom assay panel were included as model features, and a forward fitting procedure was applied to estimate which combinations of features result in better model performance. Figure 4D shows the sequential forward selection of features. A corresponding best regression plot of predicted versus actual Gd lesion counts (i.e., a proxy for MS disease activity burden in this analysis) was generated. R 2 The best performance based on is obtained using seven features (NEFL, MOG, CDCP1, OPG, APLP1, and COL4A1): 0.219 ± 0.050. Examining the Spearman r score, the best model is obtained from four features (NEFL, CDCP1, APLP1, and TNFSF13B), yielding a Spearman r score of 0.496 ± 0.038.

[0324] Example 36: Model training and validation - Prediction of annual relapse rate A logistic regression model (Elasticnet0.2) was trained and tested on 282 samples (161 low samples with ARR <0.3 and 121 high samples with ARR >0.8). Figure 5A shows the sequential forward selection of features. After progressive feature fitting and parameter hypertuning, a logistic regression classification algorithm was applied, and the best model showed an AUROC of 0.672 + / - 0.053 with the following five features: NEFL, MOG, CDCP1, IL-12B, and TNFRSF10A. Figure 5B shows the ROC curve for predicting ARR.

[0325] Example 37: Model training and validation - Prediction of clinically defined relapse A logistic regression model was constructed using the LBFGS solver with L2 regularization (C = 1.0) and balanced class weights. Relapse status was defined by clinically defined criteria in the ACP study ("worsening" vs. "static") and F6, depending on whether the patient relapsed within 90 days of blood draw. Because there were no bridging samples between ACP and F6, two independent analyses of the two studies were performed. NPX values ​​for both studies were demographically corrected, and studies in F6 where MRI was more than 30 days old were excluded (for consistency with the Gd study).

[0326] Figure 6A shows the sequential forward selection of features using the F6 study (n = 155, 136 patients without relapse and 19 with relapse in the past 90 days) and the ACP study (n = 124, 64 static MS samples and 60 worsening MS samples). As the plot moves from left to right, new features are cumulatively added to the total. Figure 6B shows the performance of each model. Specifically, the model trained on the F6 study showed an AUROC of 0.915 ± 0.053, and the model trained on the ACP study showed an AUROC of 0.845 ± 0.061.

[0327] Example 38: Model training and validation - Prediction of disease progression Figure 7 shows the sequential forward selection of features on absolute quantification data from the Expanded Disability Status Scale (EDSS). Here, biomarkers and their absolute quantification measurements were considered for 163 samples for which all data were available. The samples were from the F6 (University of Basel) research cohort. For each protein, serum measurements were directly correlated with the respective endpoint (EDSS, T2-weighted lesion volume). Only serum samples taken within 30 days of MRI were considered for the radiographic endpoint (i.e., T2-weighted lesion volume). Five-fold cross-validation was used to train and test logistic models (L2 regularization) and evaluate performance. The optimization metric used was R. 2 (Pearson's R correlation squared) and standardized coefficients are shown. The values ​​in Table 13 show the EDSS R correlation for various biomarkers.2 Of note, raw serum GFAP was 2 The raw serum OPG correlated with EDSS with an R value of 0.201. 2 It correlates with EDSS with a value of 0.204.

[0328] The values ​​shown in Table 14 represent absolute quantification (log-transformed pg / mL) versus relative quantification (normalized protein expression, or NPX) data for n=205 samples from the F6 Basel cohort. AvN1 uses Pearson's R, which corresponds to the correlation between absolute quantification and NPX values ​​measured in a test panel, to guide early research and development. 2 N2vN1 represents the same metric between the relative NPX (before a standard curve was fitted to map values ​​versus concentrations) that underlies absolute quantification and the previously measured test panel NPX. *GFAP does not have a corresponding relative quantitative measurement because a test assay did not exist at the time. This example serves to demonstrate that quantitative information from markers on a panel, regardless of source format, can be used to predict MS disease activity.

[0329] Example 39: Multivariate Biomarker Panel for Predicting Multiple Sclerosis Disease Activity (Tier A, Tier B, Tier C Biomarkers) Biomarkers are selected for multivariate custom panels according to their correlation with various endpoints (e.g., minimal disease (e.g., 0-1 Gd enhancing lesions), conventional disease (e.g., 0-at least 1 Gd enhancing lesions), annualized relapse rate, and progression vs. stasis).

[0330] Multivariate analysis of biomarker panels was performed in various human clinical studies according to the methods described in Example 3. In particular, biomarkers were classified into various tiers (e.g., Tier A, Tier B, and Tier C). Biomarker panels were constructed from one or more tiers (e.g., Tier A only, Tier B only, Tier C only, Tier A+B, or Tier A+B+C). The 21 total biomarkers evaluated in this multivariate example are as follows:

[0331] Linear regression models (L1 regularization) were trained and cross-validated on each dataset (AIM1-ARR, ACP-E vs. Q, independently (except for F4+F5, which was blended with the bridging sample through normalization for the primary endpoint Gd). When possible for the classification problem, disease activity was divided into minimal (0 vs. 1 Gd lesion), normal (0 vs. any Gd lesion), and extreme (0 vs. 3+ lesion). To report AUC / PPV, the same model building strategy was redeployed with increasingly larger subsets / tiers of markers on the panel. Statistical evaluation criteria for multivariate analysis (area under the curve (AUC) and positive predictive value (PPV)) are shown below.

[0332] In general, biomarker panels using Tier A, Tier B, and Tier C biomarkers (21 biomarkers total) represented predictive models that demonstrated improved predictive ability across various disease activity endpoints (e.g., minimal disease activity, typical disease activity, extreme disease activity, annualized relapse rate, or disease state). Tables 15A-15E show performance measures for different biomarker panels. Specifically, the AUCs across these various disease activity endpoints ranged from 0.771 to 0.961, while the PPVs ranged from 0.687 to 0.895. Biomarker panels using Tier A and B biomarkers (17 biomarkers total) achieved AUC values ​​across various disease endpoints ranging from 0.737 to 0.968, while the PPVs ranged from 0.620 to 0.896. Biomarker panels using only Tier A biomarkers (8 biomarkers total) achieved AUC values ​​across various disease endpoints ranging from 0.763 to 0.880, with PPVs ranging from 0.716 to 0.871. Biomarker panels using only Tier B biomarkers or only Tier C biomarkers maintained prediction but were significantly less predictive than biomarker panels using Tier A biomarkers or combinations of Tier A+A or Tier A+B+C. Specifically, biomarker panels using only Tier B biomarkers achieved AUC values ​​across various disease endpoints ranging from 0.562 to 0.841, with PPVs ranging from 0.462 to 0.999. Biomarker panels using only Tier C biomarkers achieved AUC values ​​across various disease endpoints ranging from 0.589 to 0.779, with PPVs ranging from 0.410 to 0.781.

[0333] Example 40: Multivariate biomarker panel for predicting multiple sclerosis disease activity Multivariate analysis of biomarker panels was performed in various human clinical studies according to the methods described in Example 3. In particular, biomarkers were classified into various tiers (e.g., Tier 1, Tier 2, and Tier 3). Biomarker panels were constructed from one or more tiers (e.g., Tier 1 only, Tier 2 only, Tier 3 only, Tier 1+2, or Tier 1+2+3). The total 21 biomarkers evaluated by this multivariate example are the 21 biomarkers shown in Table 2. Additional backup biomarkers that can be used in place of any of the 21 biomarkers are identified in Table 2 as Tier 4 biomarkers.

[0334] Linear regression models (L1 regularization) were trained and cross-validated on each dataset (AIM1-ARR, ACP-E vs. Q, independently (except for F4+F5, which was blended with bridging samples through normalization for the primary endpoint Gd)). When possible for the classification problem, disease activity was divided into minimal (0 vs. 1 Gd lesions), normal (0 vs. any Gd lesions), and extreme (0 vs. 3+ lesions). To report AUC / PPV, the same model building strategy was redeployed with increasingly larger subsets / tiers of markers on the panel. Statistical evaluation criteria for multivariate analysis (area under the curve (AUC) and positive predictive value (PPV)) are shown below.

[0335] In general, biomarker panels using Tier 1, Tier 2, and Tier 3 biomarkers (21 biomarkers total) represented predictive models that demonstrated improved predictive ability across different disease activity endpoints (e.g., minimal disease activity, usual disease activity, extreme disease activity, annualized relapse rate, or disease state). Tables 16A-16E show performance measures of different biomarker panels.

[0336] Specifically, the AUCs across these various disease activity endpoints ranged from 0.686 to 0.889, whereas the PPVs ranged from 0.648 to 0.835. Biomarker panels using tier 1 and 2 biomarkers (17 biomarkers in total) achieved AUC values ​​across various disease endpoints ranging from 0.693 to 0.892, whereas the PPVs ranged from 0.613 to 0.843. Biomarker panels using only tier 1 biomarkers (8 biomarkers in total) achieved AUC values ​​across various disease endpoints ranging from 0.667 to 0.869, whereas the PPVs ranged from 0.617 to 0.861. Biomarker panels using only tier 2 biomarkers or only tier 3 biomarkers maintained predictive potential but were significantly less predictive than biomarker panels using tier 1 biomarkers or combinations of tier 1+2 or tier 1+2+3 biomarkers. Specifically, biomarker panels using only Tier 2 biomarkers achieved AUC values ​​across various disease endpoints ranging from 0.595 to 0.761, with PPVs ranging from 0.462 to 0.769. Biomarker panels using only Tier 3 biomarkers achieved AUC values ​​across various disease endpoints ranging from 0.566 to 0.626, with PPVs ranging from 0.370 to 0.634.

[0337] Example 41: Additional multivariate (pair, triad, and quad) biomarker panels for predicting directional shifts in multiple sclerosis disease activity Multivariate analyses of minimal sets of biomarkers (e.g., pairwise, triad, and quadrant) across various human clinical studies were performed. Specifically, the minimal sets of biomarkers were analyzed for their ability to predict directional shifts in MS disease activity (e.g., increasing or decreasing disease activity (as measured by the number of gadolinium-enhancing lesions)).

[0338] To analyze the minimal predictive set of biomarkers in longitudinal patient samples, biomarker pairs, triads, and tetrad combinations were analyzed with shift (baseline-normalized, pairwise difference) analysis to predict the increase or decrease of Gd-enhancing lesions. The overall steps of the analysis are as follows: 1. Read the list of biomarkers. 2. Read these biomarker data and associated demographic and clinical characteristics for each study from the data lake. 3. All samples collected beyond a threshold time (e.g., 30 days or more) from the relevant MRI scan were excluded. 4. Calculate the difference in NPX values ​​between all pairs of samples with increasing or decreasing lesion activity. a. One sample is 0 for the true baseline normalization approach Remove only sample pairs with associated MRIs that have Gd-lesions. 5. For each study: a. Use gradual increase / decrease or Non-Gad+ → Gad+ / Gad+ → Non-Gad+ as the endpoint. b. Perform a 5-fold cross-validation split; c. Select a logistic regression model configuration that has performed well in previous multivariate analyses; d. Create feature matrices from all combinations of 2, 3, and 4 proteins (demographic / clinical characteristics are not needed as covariates as baseline normalization will factor it into the process). e. Train a copy of the logistic regression model on four of the five folds and evaluate its performance on the fifth. 6. Average the AUROC for each model across the five folds and three studies. Take the standard deviation of performance to calculate an uncertainty metric (repeated PPV). 7. Sort the models by AUROC and generate ROC curves for each study for the model corresponding to the highest performing feature set. We performed the above analysis in two ways.

[0339] The above analysis was carried out in two ways: A. Contains a complete list of biomarkers, B. Follow-up analysis excluding the best-performing biomarker (NEFL).

[0340] Tables 17 and 18 show that biomarker panels containing two, three, or four biomarkers predict a directional shift in MS disease activity.

[0341] Specifically, for biomarker pairs, a panel including the NEFL and CD6 biomarkers achieved an area under the receiver operating characteristic curve (AUROC) of 0.860 and an average PPV of 0.77. Furthermore, a biomarker panel including the NEFL and CXCL9 biomarkers achieved an AUROC of 0.85 and an average PPV of 0.74. Additional biomarker pairs (excluding NEFL) were also predictive. For example, a biomarker panel including MOG and CXCL9 achieved an AUROC of 0.761 and a PPV of 0.686. A biomarker panel including CD6 and CXCL9 achieved an AUROC of 0.766 and a PPV of 0.699. A biomarker panel including MOG and CD6 achieved an AUROC of 0.745 and a PPV of 0.705.

[0342] In particular, for biomarker triads, a panel including NEFL, CXCL9, and CD6 achieved an AUROC of 0.885 and a mean PPV of 0.79. Additionally, a biomarker panel including MOG, CD6, and CXCL9 achieved an AUROC of 0.798 and a PPV of 0.71.

[0343] Specifically, for biomarker quads, a biomarker panel including NEFL, MOG, CXCL9, and CD6 achieved an AUROC of 0.884 and a PPV of 0.763. A biomarker panel including NEFL, CXCL9, CD6, and CXCL13 achieved an AUROC of 0.889 and a PPV of 0.764. A biomarker panel including NEFL, TNFRSF10A, COL4A1, and CCL20 achieved an AUROC of 0.836 and a PPV of 0.725. Additional biomarker quads (excluding NEFL) also demonstrated predictive potential. For example, the combination of MOG, CXCL9, IL-12B, and APLP1 achieved an AUROC of 0.795 and a PPV of 0.67. The combination of CXCL9, OPG, APLP1, and OPN achieved an AUROC of 0.741 and a PPV of 0.65. The combination of MOG, IL-12B, OPN, and CNTN2 achieved an AUROC of 0.765 and a PPV of 0.69.

[0344] Example 42: Additional multivariate (paired, triad, and quad) biomarker panels for predicting the presence or absence of multiple sclerosis Multivariate analyses of minimal sets of biomarkers (e.g., pairwise, triad, and quadrant) across various human clinical studies were performed. Specifically, the minimal sets of biomarkers were analyzed for their ability to predict classification of usual MS disease presence or absence (e.g., presence indicated by at least one Gd-enhancing lesion and absence indicated by zero Gd-enhancing lesions).

[0345] To analyze the minimal predictive set of biomarkers, biomarker pairs, triads, and quadrant combinations were analyzed in a cross-sectional analysis to predict the presence or absence of Gd-enhancing lesions ("normal disease activity" or GDA). The overall steps of the analysis were as follows: 1. Read the list of biomarkers. 2. For each study, data on these biomarkers and associated demographic and clinical characteristics will be read from the data lake. 3. Exclude all samples collected beyond a threshold time (e.g., more than 30 days) from the relevant MRI scan. 4. Implement optimized demographic and clinical adjustments. A. Using only the Gd samples (without any disease activity) from a given study, OLS regressions are performed between the optimal subset of demographic / clinical characteristics and the NPX levels of individual biomarker values, excluding outliers. a. Disease duration: number of years since MS diagnosis. b. Age: The patient's age in years. c. Sample age: how long the sample was stored before being formally analyzed. d. Delta_BloodMinusDiagnosis: Number of days between MRI and each blood sample (must be within 30 days). B. Extract the residuals from this procedure and use them as input to the model building procedure. 5. For each study: a. Develop GDA evaluation items, b. Perform a 5-fold cross-validation split; c. Select a logistic regression model configuration that has performed well in previous multivariate analyses; d. Create feature matrices for all combinations of two, three, and four proteins. (without any demographic / clinical characteristics as covariates), e. Train a copy of the logistic regression model on four of the five folds, The fifth step is to evaluate the performance. 6. Average the AUROC for each model across the five folds and three studies. 7. Sort the models by AUROC and generate ROC curves for each study for the model corresponding to the highest performing feature set.

[0346] The above analysis was carried out in two ways: A. Contains a complete list of biomarkers, B. Follow-up analysis excluding the best-performing biomarker (NEFL).

[0347] Table 19 below shows that biomarker panels containing two, three, or four biomarkers predict the presence or absence of MS. In particular, for biomarker pairs, a panel including NEFL and TNFSF13B achieved an AUROC of 0.788 and a PPV of 0.708, a panel including NEFL and CNTN2 achieved an AUROC of 0.777 and a PPV of 0.732, and a panel including NEFL and CXCL9 achieved an AUROC of 0.777 and a PPV of 0.713. Additional biomarker pairs (without NEFL) also predicted the presence or absence of multiple sclerosis. The panel including MOG and CDCP1 achieved an AUROC of 0.672 and a PPV of 0.597, the panel including MOG and TNFSF13B achieved an AUROC of 0.672 and a PPV of 0.599, and the panel including MOG and CXCL9 achieved an AUROC of 0.670 and a PPV of 0.606.

[0348] Specifically, for the biomarker triad, a panel including NEFL, CNTN2, and TNFSF13B achieved an AUROC of 0.794 and a PPV of 0.745. Substituting either APLP1 or TNFRSF10A for CNTN2 in the biomarker triad achieved similar AUROC values ​​of 0.794 and 0.792, respectively. For the biomarker triad without NEFL, a panel including MOG, CXCL9, and TNFSF13B achieved an AUROC of 0.690 and a PPV of 0.623; a panel including MOG, OPG, and TNFSF13B achieved an AUROC of 0.685 and a PPV of 0.637; and a panel including MOG, CCL20, and TNFSF13B achieved an AUROC of 0.685 and a PPV of 0.664. APLP1, CCL20, and CNTN2 were the next proteins that helped improve the signal beyond the biomarker triad. For the biomarker quad, a panel including NEFL, TNFRSF10A, CNTN2, and TNFSF13B achieved an AUROC of 0.798 and a PPV of 0.742.

[0349] Example 43: Additional multivariate (paired, triad, and quad) biomarker panels for predicting multiple sclerosis severity Multivariate analyses of minimal sets of biomarkers (e.g., pairwise, triad, and tetrad) were performed across various human clinical studies according to the methods described in Example 3. Specifically, the minimal sets of biomarkers were analyzed for their ability to predict Gd-enhancing lesion counts (e.g., minimal MS disease measures).

[0350] Regression analysis, as well as cross-sectional and shift analysis, was conducted using the following procedure. 1. Read the list of biomarkers. 2. Read data on these biomarkers and associated demographic and clinical characteristics for each study from the data lake. 3. All samples collected beyond a threshold time (e.g., 30 days or more) from the relevant MRI scan were excluded. 4. Implement optimized demographic and clinical adjustments. A. Using only the Gd samples (without any disease activity) from a given study, OLS regressions are performed between the optimal subset of demographic / clinical characteristics and the NPX levels of individual biomarker values, excluding outliers. a. Disease duration: number of years since MS diagnosis. b. Age: The patient's age in years. c. Sample age: how long the sample was stored before being formally analyzed. d. Delta_BloodMinusDiagnosis: Number of days between MRI and each blood sample (must be within 30 days). B. Extract the residuals from this procedure and use them as input to the model building procedure. 5. For each study: a. Gd lesion count (maximum clipped at 5) was used as the regression endpoint. b. Perform a 5-fold cross-validation split; c. Select a ridge regression model configuration that has performed well in previous multivariate analyses; d. Create feature matrices from all combinations of two, three, and four proteins. (without any demographic / clinical characteristics as covariates), e. Train a copy of the ridge regression model on four of the five folds, The fifth step is to evaluate the performance. 6. Adjusted R for each model across five folds and three studies 2 Average the values. 7. Adjusted model R 2 and create a correlation plot between prediction and endpoint for each study of the model corresponding to the best performing feature set.

[0351] The above analysis was carried out in two ways: A. Contains a complete list of biomarkers, B. Follow-up analysis excluding the best-performing biomarker (NEFL).

[0352] Table 20 below shows that biomarker panels containing two, three, or four biomarkers are generally predictive for determining minimal MS disease activity.

[0353] Specifically, for biomarker pairs, a panel including NEFL and TNFSF13B achieved a Spearman's R coefficient value of 0.524, a panel including NEFL and SERPINA9 achieved a Spearman's R coefficient value of 0.501, and a panel including NEFL and GH achieved a Spearman's R coefficient value of 0.505. Additional biomarker pairs (without NEFL) were also used to assess minimal MS disease activity. For example, a panel including MOG and TNFSF13B achieved a Spearman's R coefficient value of 0.286, and a panel including MOG and CXCL9 achieved a Spearman's R coefficient value of 0.290.

[0354] For biomarker triads, a panel including NEFL, SERPINA9, and TNFSF13B achieved a Spearman's R coefficient value of 0.533, a panel including NEFL, CNTN2, and TNFSF13B achieved a Spearman's R coefficient value of 0.525, a panel including NEFL, APLP1, and TNFSF13B achieved a Spearman's R coefficient value of 0.537, a panel including MOG, CXCL9, and TNFSF13B achieved a Spearman's R coefficient value of 0.306, a panel including MOG, CCL20, and COL4A1 achieved a Spearman's R coefficient value of 0.210, and a panel including CXCL13, APLP1, and FLRT2 achieved a Spearman's R coefficient value of 0.143.

[0355] For biomarker quartets, a panel including NEFL, CXCL13, CCL20, and TNFSF13B achieved a Spearman's R coefficient value of 0.520. Additionally, a panel including NEFL, CXCL13, CXCL9, and TNFSF13B achieved a Spearman's R coefficient value of 0.513, and a panel including NEFL, CXCL13, SERPINA9, and TNFSF13B achieved a Spearman's R coefficient value of 0.513. The biomarker quartet of MOG, CXCL9, OPG, SERPINA9, and TNFSF13B (excluding NEFL) achieved a Spearman's R coefficient value of 0.302.

[0356] Example 44: Multivariate (paired, triad, and quad) biomarker panels for predicting multiple sclerosis disease progression Multivariate analysis of minimal sets of biomarkers (e.g., pairwise, triad, and quadrant) was performed on n=205 samples from the University Hospital of Basel according to the methods described in Example 3. Specifically, minimal sets of biomarkers were analyzed for their ability to predict primary disease endpoints of disease progression (e.g., association of serum protein measurements with the Expanded Disability Status Scale (EDSS)). All sets use mathematical combinations (e.g., logistic regression models or decision trees) to combine individual biomarker levels into a multivariate score.

[0357] Ridge (L2) regularized linear models were evaluated with 5-fold cross-validation (estimated means and uncertainties) across all exhaustive pairwise, triplet, and quadruplet combinations of protein subsets. Performance was then sorted by Spearman R2 (per the rationale above). This procedure was repeated for: 1. Model constructed using only logarithmic transformation of protein concentrations 2. Models built with logarithmic transformation of protein concentrations and covariates of age, sex, and disease duration incorporated as eligible features. 3. To predict each biomarker concentration from only the demographic information (age, sex, and disease duration) contained in samples with no disease activity (i.e., 0 Gd lesions on the corresponding MRI), a model was built with a logarithmic transformation of the protein concentrations after the residuals were extracted from the OLS regression procedure.

[0358] Table 21 documents the best-performing minimal predictive biomarker sets (e.g., pairs, triads, and quads) according to EDSS regression to predict lesion counts based on protein signatures in serum with associated MRI at a single blood draw within 30 days of MRI. Notably, the biomarker triad of GFAP, NEFL, and MOG had the overall highest adjusted R 2 (with measurable improvement over the best univariate feature in the field - GFAP). Furthermore, the biomarker pairs A) GFAP and MOG, B) GFAP and NEFL, and C) APLP1 and GFAP, as well as the biomarker tetrad GFAP, NEFL, MOG, and IL-12B / PRTG / APLP1, also showed predictive potential.

[0359] The best biomarker set, including covariate (age, sex, disease duration) adjusted Log(pg / mL), had the highest adjusted R 2The biomarker pairs include NEFL and MOG, which have been shown to predict EDSS in an improved manner. The biomarker triads NEFL, MOG, and GH, and NEFL, MOG, and SERPINA9 also predict EDSS in an improved manner. The biomarker quads NEFL, MOG, GH, and SERPINA9, and NEFL, MOG, GH, and TNFRSF10A are also further improved. Additionally, the biomarker triads CXCL9, OPG, and SERPINA9 are predictive of disease progression, and are further improved when MOG is added as a biomarker quad.

[0360] The best biomarker set with adjusted demographics (age, sex, disease duration) included the biomarker pair CXCL9 and OPG, and the biomarker triad CXCL9, OPG, and TNFRSF10A. The biomarker tetrad CD6, IL-12B, APLP1, and CCL20 had the highest Pearson R of any demographically adjusted model. 2 Form correlations.

[0361] Example 45: Biomarker panel for predicting disease progression (brain parenchymal fraction) Univariate and multivariate biomarker panels were constructed to predict brain parenchymal fraction (BPF), which is known to be associated with MS disease progression. The biomarker panel was constructed and analyzed using the F6 study (University Hospital Basel Cohort (UHBC)). The cohort included 205 samples from 88 patients (67 women and 21 men) at the University Hospital Basel. Samples were collected using paired MRI scans between July 2012 and August 2019. Patients had two to six longitudinal samples (all patients had Gd-enhancing lesions in at least one sample and none in the other). The cohort was selected to be balanced across age, sex, disease duration, and EDSS for a study in which the primary endpoint was Gd-lesion counts. Characteristics of the UHBC cohort are shown in Table 22.

[0362] Patient images were analyzed and labeled. Specifically, 3D T1 and FLAIR images were uploaded to the Octave cloud environment, and quality control of the raw data was performed by two experienced raters. Data quality was assessed on a scale of 1 to 5 (1 = poor, 3 = average, 5 = excellent). Image segmentation of T1 and FLAIR images was performed via Cortechs' FDA-approved LesionQuant software. A second quality control of the segmentation was performed by the raters.

[0363] Univariate biomarker panels were constructed using individual biomarkers se...

Claims

1. 1. A method for predicting multiple sclerosis disease progression in a subject, said method comprising: obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises one or more biomarkers from at least one group selected from Group 1, Group 2, and Group 3; Group 1 includes one or more of biomarker 1, biomarker 2, biomarker 3, biomarker 4, biomarker 5, biomarker 6, biomarker 7, and biomarker 8; Biomarker 1 is GFAP, NEFL, OPN, CXCL9, MOG, or CHI3L1; Biomarker 2 is CDCP1, IL-18BP, IL-18, GFAP, or MSR1; Biomarker 3 is MOG, CADM3, KLK6, BCAN, OMG, or GFAP; Biomarker 4 is CXCL13, NOS3, or MMP-2; Biomarker 5 is OPG, TFF3, or ENPP2; Biomarker 6 is APLP1, SEZ6L, BCAN, DPP6, NCAN, or KLK6; Biomarker 7 is VCAN, TINAGL1, CANT1, NECTIN2, MMP-9, or NPDC1; Biomarker 8 is NEFL, MOG, CADM3, or GFAP, and Group 2 includes one or more of biomarker 9, biomarker 10, biomarker 11, biomarker 12, biomarker 13, biomarker 14, biomarker 15, biomarker 16, and biomarker 17; Biomarker 9 is CXCL9, CXCL10, IL-12B, CXCL11, or GFAP; Biomarker 10 is TNFRSF10A, TNFRSF11A, SPON2, CHI3L1, or IFI30; Biomarker 11 is CCL20, CCL3, or TWEAK; Biomarker 12 is TNFSF13B, CXCL16, ALCAM, or IL-18; Biomarker 13 is OPN, OMD, MEPE, or GFAP; Biomarker 14 is SERPINA9, TNFRSF9, or CNTN4; Biomarker 15 is CD6, CD5, CRTAM, CD244, or TNFRSF9; Biomarker 16 is FLRT2, DDR1, NTRK2, CDH6, MMP-2; Biomarker 17 is CNTN2, DPP6, GDNFR-α-3, or SCARF2, and Group 3 includes one or more of biomarker 18, biomarker 19, biomarker 20, and biomarker 21; Biomarker 18 is COL4A1, IL6, Notch3, or PCDH17; Biomarker 19 is GH, GH2, or IGFBP-1; Biomarker 20 is IL-12B, IL12A, or CXCL9, and Biomarker 21 is PRTG, NTRK2, NTRK3, or CNTN4; And, generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; The method comprising:

2. 2. The method of claim 1, wherein the plurality of biomarkers comprises each biomarker in Group 1, wherein biomarker 1 is GFAP, biomarker 2 is CDCP1, biomarker 3 is MOG, biomarker 4 is CXCL13, biomarker 5 is OPG, biomarker 6 is APLP1, biomarker 7 is VCAN, and biomarker 8 is NEFL.

3. The performance of the predictive model has a correlation coefficient (R ２ 3. The method of claim 2, characterized by:

4. 3. The method of claim 2, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

77.

5. 3. The method of claim 2, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

6. 3. The method of claim 2, wherein the plurality of biomarkers further comprises each biomarker of Group 2, wherein biomarker 9 is CXCL9, biomarker 10 is TNFRSF10A, biomarker 11 is CCL20, biomarker 12 is TNFSF13B, biomarker 13 is OPN, biomarker 14 is SERPINA9, biomarker 15 is CD6, biomarker 16 is FLRT, and biomarker 17 is CNTN2.

7. The performance of the predictive model has a correlation coefficient (R ２ 7. The method of claim 6, characterized by:

8. 7. The method of claim 6, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

76.

9. 7. The method of claim 6, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

10. 10. The method of any one of claims 6 to 9, wherein the plurality of biomarkers further comprises each biomarker of Group 3, wherein biomarker 18 is COL4A1, biomarker 19 is GH, biomarker 20 is IL-12B, and biomarker 21 is PRTG.

11. The performance of the predictive model has a correlation coefficient (R ２ 11. The method of claim 10, characterized by:

12. 11. The method of claim 10, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

13. 11. The method of claim 10, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

14. 2. The method of claim 1, wherein the plurality of biomarkers comprises one or more biomarkers of Group 1, and the one or more biomarkers of Group 1 comprises GFAP.

15. 15. The method of claim 14, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

16. 16. The method of claim 14 or 15, wherein the performance of the predictive model is characterized by an AUROC between 0.72 and 0.

78.

17. The performance of the predictive model is at least Pearson's R of 0.

10. ２ The method of claim 14 characterized by a coefficient.

18. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ 16. The method of claim 14 or 15, characterized by a coefficient.

19. The method of claim 1 , wherein the plurality of biomarkers does not include GFAP.

20. 20. The method of claim 19, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

21. 21. The method of claim 19 or 20, wherein the performance of the predictive model is characterized by an AUROC between 0.66 and 0.

70.

22. The performance of the predictive model is at least Pearson's R of 0.

10. ２ The method of claim 14 characterized by a coefficient.

23. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 16. The method of claim 14 or 15, characterized by a coefficient.

24. The method according to any one of claims 1 to 23, wherein the prediction of multiple sclerosis disease progression is based on an evaluation criterion called brain parenchymal fraction value.

25. The method according to any one of claims 1 to 23, wherein the prediction of multiple sclerosis disease progression is based on an evaluation criterion called the Expanded Disability Status Scale (EDSS) score.

26. 26. The method of claim 25, wherein an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6 or greater indicates severe MS disease progression.

27. The method according to any one of claims 1 to 23, wherein the prediction of multiple sclerosis disease progression is based on an evaluation criterion called the Patient-Determined Disease Stage (PDDS) score.

28. 28. The method of claim 27, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe and mild / moderate MS disease progression.

29. 29. The method of claim 28, wherein a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

30. The method of claims 1 to 23, wherein the prediction of multiple sclerosis disease progression is the PRO Measurement Information System (PROMIS) score.

31. The method according to claims 1 to 23, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

32. 1. A method for predicting multiple sclerosis disease progression in a subject, said method comprising: below: one or more neuroaxonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immune-modulating biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; or one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN. obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers including at least one of: generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; The method comprising:

33. 1. A method for predicting multiple sclerosis disease progression in a subject, said method comprising: below: one or more neuroaxonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immune-modulating biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN; or one or more cerebrovascular function biomarkers selected from the group consisting of COL4A1, VCAN, GFAP, and CD6 obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers including at least one of: generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; The method comprising:

34. 34. The method of claim 32 or 33, wherein the one or more axonal integrity biomarkers include NEFL, OPG, and GFAP, the one or more neuroinflammation biomarkers include CXCL13 and CXCL9, the one or more immunomodulatory biomarkers include CDCP1, and the one or more myelination biomarkers include MOG and APLP1.

35. 35. The method of claim 34, wherein the one or more axonal integrity biomarkers further comprise SERPINA9, FLRT2, and CNTN2; the one or more neuroinflammation biomarkers further comprise CCL20, CXCL9, TNFRSF10A, and CD6; the one or more immune modulation biomarkers further comprise TNFSF13B; and the one or more myelination biomarkers further comprise OPN.

36. 36. The method of claim 35, wherein the one or more neuroaxonal integrity biomarkers further comprise PRTG and the one or more immunomodulatory biomarkers further comprise IL-12B.

37. The performance of the predictive model has a correlation coefficient (R ２ 37. The method of claim 36, characterized by:

38. 37. The method of claim 36, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

39. 37. The method of claim 36, wherein the performance of the predictive model is characterized by a PPV of at least 0.

17.

40. 33. The method of claim 32, wherein the plurality of biomarkers comprises one or more axon integrity biomarkers, and wherein the one or more axon integrity biomarkers comprises GFAP.

41. 41. The method of claim 40, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

42. 42. The method of claim 40 or 41, wherein the performance of the predictive model is characterized by an AUROC between 0.72 and 0.

78.

43. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 41. The method of claim 40 characterized by a coefficient.

44. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ 42. The method of claim 40 or 41, characterized by a coefficient.

45. 33. The method of claim 32, wherein the plurality of biomarkers does not include GFAP.

46. 46. ​​The method of claim 45, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

47. 47. The method of claim 45 or 46, wherein the performance of the predictive model is characterized by an AUROC between 0.66 and 0.

70.

48. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 46. ​​The method of claim 45, characterized by a coefficient.

49. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 47. The method of claim 45 or 46, characterized by a coefficient.

50. The method of any one of claims 32 to 49, wherein the prediction of multiple sclerosis disease progression is a brain parenchymal fraction value.

51. 50. The method of any one of claims 32 to 49, wherein the prediction of multiple sclerosis disease progression is the Expanded Disability Status Scale (EDSS) score.

52. 52. The method of claim 51, wherein an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6.0 or greater indicates severe MS disease progression.

53. 50. The method of any one of claims 32 to 49, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score.

54. 54. The method of claim 53, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe and mild / moderate MS disease progression.

55. 55. The method of claim 54, wherein a Patient Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

56. 50. The method of any one of claims 32 to 49, wherein the prediction of multiple sclerosis disease progression is the PRO Measurement Information System (PROMIS) score.

57. The method of claims 32 to 49, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

58. 1. A method for predicting multiple sclerosis disease progression in a subject, said method comprising: obtaining, or having obtained, a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises two or more of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG; generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; The method comprising:

59. 59. The method of claim 58, wherein the plurality of biomarkers comprises each of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG.

60. The performance of the predictive model has a correlation coefficient (R ２ 60. The method of claim 59, characterized by:

61. 60. The method of claim 59, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

62. 60. The method of claim 59, wherein the performance of the predictive model is characterized by a PPV of at least 0.

17.

63. 59. The method of claim 58, wherein the plurality of biomarkers comprises GFAP.

64. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1.

65. 64. The method of claim 63, wherein the plurality of biomarkers further comprises APLP1.

66. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL13.

67. 64. The method of claim 63, wherein the plurality of biomarkers further comprises MOG.

68. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG.

69. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1 and APLP1.

70. 64. The method of claim 63, wherein the plurality of biomarkers further comprises MOG and CDCP1.

71. 64. The method of claim 63, wherein the plurality of biomarkers further comprises APLP1 and CXCL13.

72. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1 and SERPINA9.

73. 64. The method of claim 63, wherein the plurality of biomarkers further comprises MOG and CXCL13.

74. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1, CCL20, and APLP1.

75. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1, APLP1, and CXCL13.

76. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1, CCL20, and MOG.

77. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1, APLP1, and SERPINA9.

78. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1, MOG, and APLP1.

79. 79. The method of any one of claims 63 to 78, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

80. 80. The method of any one of claims 63 to 79, wherein the performance of the predictive model is characterized by an AUROC of between 0.72 and 0.

78.

81. 64. The method of claim 63, wherein the plurality of biomarkers further comprises MOG.

82. 64. The method of claim 63, wherein the plurality of biomarkers further comprises APLP1.

83. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG.

84. 64. The method of claim 63, wherein the plurality of biomarkers further comprises TNFRSF10A.

85. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CDCP1.

86. 64. The method of claim 63, wherein the plurality of biomarkers further comprises APLP1.

87. 64. The method of claim 63, wherein the plurality of biomarkers further comprises NEFL.

88. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CNTN2.

89. 64. The method of claim 63, wherein the plurality of biomarkers further comprises GH.

90. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9.

91. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG and MOG.

92. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG and APLP1.

93. 64. The method of claim 63, wherein the plurality of biomarkers further comprises TNFRSF10A and MOG.

94. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9 and OPG.

95. 64. The method of claim 63, wherein the plurality of biomarkers further comprises TNFRSF10A and APLP1.

96. 64. The method of claim 63, wherein the plurality of biomarkers further comprises APLP1 and NEFL.

97. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL13 and APLP1.

98. 64. The method of claim 63, wherein the plurality of biomarkers further comprises FLRT2 and APLP1.

99. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9 and APLP1.

100. 64. The method of claim 63, wherein the plurality of biomarkers further comprises GH and APLP1.

101. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9, OPG, and MOG.

102. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CNTN2, OPG, and MOG.

103. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9, OPG, and APLP1.

104. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG, PRTG, and MOG.

105. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPG, OPN, and MOG.

106. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL13, APLP1, and NEFL.

107. 64. The method of claim 63, wherein the plurality of biomarkers further comprises FLRT2, APLP1, and NEFL.

108. 64. The method of claim 63, wherein the plurality of biomarkers further comprises OPN, APLP1, and NEFL.

109. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL9, APLP1, and NEFL.

110. 64. The method of claim 63, wherein the plurality of biomarkers further comprises CXCL13, FLRT2, and APLP1.

111. The performance of the predictive model is at least Pearson's R of 0.

10. ２ The method according to any one of claims 81 to 110, characterized by a coefficient.

112. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ The method according to any one of claims 81 to 111, characterized by a coefficient.

113. 59. The method of claim 58, wherein said plurality of biomarkers does not include GFAP.

114. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1 and OPG.

115. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1 and SERPINA9.

116. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and TNFRSF10A.

117. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and MOG.

118. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1 and MOG.

119. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, MOG, and OPG.

120. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, SERPINA9, and OPG.

121. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, OPG, and CXCL13.

122. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, CXCL9, and OPG.

123. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, FLRT2, and OPG.

124. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, MOG, OPG, and CXCL13.

125. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, MOG, TNFRSF10A, and OPG.

126. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, CXCL9, SERPINA9 and OPG.

127. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, CNTN2, SERPINA9, and OPG.

128. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, SERPINA9, CD6, and OPG.

129. 129. The method of any one of claims 113 to 128, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

130. 130. The method of any one of claims 113 to 129, wherein the performance of the predictive model is characterized by an AUROC of between 0.66 and 0.

70.

131. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and NEFL.

132. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and OPN.

133. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and FLRT2.

134. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG and MOG.

135. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL9 and OPG.

136. 114. The method of claim 113, wherein the plurality of biomarkers comprises GH and NEFL.

137. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL13 and NEFL.

138. 114. The method of claim 113, wherein the plurality of biomarkers comprises APLP1 and NEFL.

139. 114. The method of claim 113, wherein the plurality of biomarkers comprises CCL20 and NEFL.

140. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL9 and NEFL.

141. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG, MOG, and NEFL.

142. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG, FLRT2, and NEFL.

143. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL9, OPG, and NEFL.

144. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG, CDCP1, and NEFL.

145. 114. The method of claim 113, wherein the plurality of biomarkers comprises OPG, OPN, and NEFL.

146. 114. The method of claim 113, wherein the plurality of biomarkers comprises GH, APLP1, and NEFL.

147. 114. The method of claim 113, wherein the plurality of biomarkers comprises GH, CXCL13, and NEFL.

148. 114. The method of claim 113, wherein the plurality of biomarkers comprises GH, CDCP1, and NEFL.

149. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL13, CCL20, and NEFL.

150. 114. The method of claim 113, wherein the plurality of biomarkers comprises GH, CCL20, and NEFL.

151. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL.

152. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13 and NEFL.

153. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL.

154. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL.

155. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and MOG.

156. 114. The method of claim 113, wherein the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL.

157. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL.

158. 114. The method of claim 113, wherein the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL.

159. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13 and NEFL.

160. 114. The method of claim 113, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and MOG.

161. The performance of the predictive model is at least Pearson's R of 0.

10. ２ The method of any one of claims 131 to 160, characterized by a coefficient.

162. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 162. The method of any one of claims 131 to 161, characterized by a coefficient.

163. The method of any one of claims 58 to 162, wherein the prediction of multiple sclerosis disease progression is a brain parenchymal fraction value.

164. 163. The method of any one of claims 58 to 162, wherein the prediction of multiple sclerosis disease progression is the Expanded Disability Status Scale (EDSS) score.

165. 165. The method of claim 164, wherein an Expanded Disability Status Scale (EDSS) score of 6 or less indicates mild / moderate MS disease progression, and an EDSS score of greater than 6.5 indicates severe MS disease progression.

166. 163. The method of any one of claims 58 to 162, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score.

167. 167. The method of claim 166, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression.

168. 168. The method of claim 167, wherein a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

169. 163. The method of claims 58 to 162, wherein the prediction of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

170. 163. The method of any one of claims 58 to 162, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

171. 171. The method of any one of claims 1 to 170, wherein generating a prediction of multiple sclerosis disease progression by applying the predictive model to expression levels of a plurality of biomarkers further comprises applying the predictive model to one or more subject attributes of the subject, wherein the subject attributes include any of age, sex, and disease duration.

172. 172. The method of any one of claims 1 to 171, wherein generating the prediction of multiple sclerosis disease progression comprises comparing the score output by the predictive model to a reference score.

173. The reference score is A) EDSS score; B) Brain parenchymal fraction value; C) PDDS score; D) PROMIS score; or E) MSRS-R score 173. The method of claim 172, corresponding to any one of the following:

174. 174. The method of claim 173, wherein the reference score further corresponds to mild / moderate MS disease progression or severe MS disease progression.

175. 175. The method of any one of claims 1-174, wherein the expression levels of the plurality of biomarkers are determined from a test sample obtained from the subject.

176. 176. The method of claim 175, wherein the test sample is a blood or serum sample.

177. 177. The method of claim 175 or 176, wherein the subject has, is suspected of having, or has previously been diagnosed with multiple sclerosis.

178. 178. The method of any one of claims 1-177, wherein obtaining or having obtained said dataset comprises performing an immunoassay to determine said expression levels of said plurality of biomarkers.

179. 179. The method of claim 178, wherein the immunoassay is a proximity extension assay (PEA) or a LUMINEX xMAP multiplex assay.

180. 180. The method of claim 178 or 179, wherein performing the immunoassay comprises contacting the test sample with a plurality of reagents comprising antibodies.

181. 181. The method of claim 180, wherein the antibody comprises one of a monoclonal antibody and a polyclonal antibody.

182. 181. The method of claim 180, wherein the antibodies include both monoclonal and polyclonal antibodies.

183. selecting a therapy for administration to said subject based on said prediction of multiple sclerosis disease progression.

183. The method of any one of claims 1 to 182, further comprising:

184. determining the therapeutic efficacy of a therapy previously administered to said subject based on said prediction of multiple sclerosis disease progression.

183. The method of any one of claims 1 to 182, further comprising:

185. 185. The method of claim 184, wherein determining the therapeutic efficacy of the treatment comprises comparing the prediction to a previous prediction determined for the subject at a previous time point.

186. 186. The method of claim 185, wherein determining the therapeutic efficacy of the treatment comprises determining that the treatment is demonstrating efficacy depending on a difference between the prediction and the previous prediction.

187. 186. The method of claim 185, wherein determining the therapeutic efficacy of the treatment comprises determining that the treatment lacks efficacy in response to a lack of difference between the prediction and the previous prediction.

188. 1. A non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: obtaining a dataset comprising expression levels of a plurality of biomarkers, wherein the plurality of biomarkers comprises one or more biomarkers from at least one group selected from Group 1, Group 2, and Group 3; Group 1 includes one or more of biomarker 1, biomarker 2, biomarker 3, biomarker 4, biomarker 5, biomarker 6, biomarker 7, and biomarker 8; Biomarker 1 is GFAP, NEFL, OPN, CXCL9, MOG, or CHI3L1; Biomarker 2 is CDCP1, IL-18BP, IL-18, GFAP, or MSR1; Biomarker 3 is MOG, CADM3, KLK6, BCAN, OMG, or GFAP; Biomarker 4 is CXCL13, NOS3, or MMP-2; Biomarker 5 is OPG, TFF3, or ENPP2; Biomarker 6 is APLP1, SEZ6L, BCAN, DPP6, NCAN, or KLK6; Biomarker 7 is VCAN, TINAGL1, CANT1, NECTIN2, MMP-9, or NPDC1; Biomarker 8 is NEFL, MOG, CADM3, or GFAP, and Group 2 includes one or more of biomarker 9, biomarker 10, biomarker 11, biomarker 12, biomarker 13, biomarker 14, biomarker 15, biomarker 16, and biomarker 17; Biomarker 9 is CXCL9, CXCL10, IL-12B, CXCL11, or GFAP; Biomarker 10 is TNFRSF10A, TNFRSF11A, SPON2, CHI3L1, or IFI30; Biomarker 11 is CCL20, CCL3, or TWEAK; Biomarker 12 is TNFSF13B, CXCL16, ALCAM, or IL-18; Biomarker 13 is OPN, OMD, MEPE, or GFAP; Biomarker 14 is SERPINA9, TNFRSF9, or CNTN4; Biomarker 15 is CD6, CD5, CRTAM, CD244, or TNFRSF9; Biomarker 16 is FLRT2, DDR1, NTRK2, CDH6, MMP-2; Biomarker 17 is CNTN2, DPP6, GDNFR-α-3, or SCARF2, and Group 3 includes one or more of biomarker 18, biomarker 19, biomarker 20, and biomarker 21; Biomarker 18 is COL4A1, IL-6, Notch3, or PCDH17; Biomarker 19 is GH, GH2, or IGFBP-1; Biomarker 20 is IL-12B, IL12A, or CXCL9, and Biomarker 21 is PRTG, NTRK2, NTRK3, or CNTN4; And, generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; An order to perform The non-transitory computer-readable medium comprising:

189. The non-transitory computer-readable medium of claim 188, wherein the plurality of biomarkers comprises each biomarker of Group 1, wherein biomarker 1 is GFAP, biomarker 2 is CDCP1, biomarker 3 is MOG, biomarker 4 is CXCL13, biomarker 5 is OPG, biomarker 6 is APLP1, biomarker 7 is VCAN, and biomarker 8 is NEFL.

190. The performance of the predictive model has a correlation coefficient (R ２ 190. The non-transitory computer-readable medium of claim 189, characterized by:

191. 190. The non-transitory computer-readable medium of claim 189, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

77.

192. 190. The non-transitory computer-readable medium of claim 189, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

193. 190. The non-transitory computer readable medium of claim 189, wherein the plurality of biomarkers further comprises each biomarker of Group 2, wherein biomarker 9 is CXCL9, biomarker 10 is TNFRSF10A, biomarker 11 is CCL20, biomarker 12 is TNFSF13B, biomarker 13 is OPN, biomarker 14 is SERPINA9, biomarker 15 is CD6, biomarker 16 is FLRT, and biomarker 17 is CNTN2.

194. The performance of the predictive model has a correlation coefficient (R ２ 194. The non-transitory computer-readable medium of claim 193, characterized by:

195. 194. The non-transitory computer-readable medium of claim 193, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

76.

196. 200. The non-transitory computer-readable medium of claim 193, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

197. 197. The non-transitory computer readable medium of any one of claims 193-196, wherein the plurality of biomarkers further comprises each biomarker of Group 3, wherein biomarker 18 is COL4A1, biomarker 19 is GH, biomarker 20 is IL-12B, and biomarker 21 is PRTG.

198. The performance of the predictive model has a correlation coefficient (R ２ 200. The non-transitory computer-readable medium of claim 197, characterized by:

199. 200. The non-transitory computer-readable medium of claim 197, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

200. 200. The non-transitory computer-readable medium of claim 197, wherein the performance of the predictive model is characterized by a PPV of at least 0.

19.

201. 189. The non-transitory computer-readable medium of claim 188, wherein the plurality of biomarkers comprises one or more biomarkers of Group 1, and wherein the one or more biomarkers of Group 1 comprises GFAP.

202. 202. The non-transitory computer-readable medium of claim 201, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

203. 203. The non-transitory computer-readable medium of claim 201 or 202, wherein the performance of the predictive model is characterized by an AUROC between 0.72 and 0.

78.

204. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 202. The non-transitory computer-readable medium of claim 201 characterized by a coefficient.

205. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ 203. The non-transitory computer-readable medium of claim 201 or 202, characterized by a coefficient.

206. The non-transitory computer-readable medium of claim 188, wherein said plurality of biomarkers does not include GFAP.

207. 207. The non-transitory computer-readable medium of claim 206, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

208. 208. The non-transitory computer-readable medium of claim 206 or 207, wherein the performance of the predictive model is characterized by an AUROC of between 0.66 and 0.

70.

209. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 207. The non-transitory computer-readable medium of claim 206, characterized by a coefficient.

210. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 210. The non-transitory computer-readable medium of claim 206 or 209, characterized by a coefficient.

211. The non-transitory computer readable medium of any one of claims 188 to 210, wherein the prediction of multiple sclerosis disease progression is based on a criterion called brain parenchymal fraction value.

212. The non-transitory computer-readable medium of any one of claims 188 to 210, wherein the prediction of multiple sclerosis disease progression is based on an evaluation criterion of Expanded Disability Status Scale (EDSS) score.

213. 213. The non-transitory computer-readable medium of claim 212, wherein an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression and an EDSS score of 6 or greater indicates severe MS disease progression.

214. The non-transitory computer-readable medium of any one of claims 188 to 210, wherein the prediction of multiple sclerosis disease progression is an evaluation criterion called the Patient-Determined Disease Stage (PDDS) score.

215. The non-transitory computer readable medium of claim 214, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression.

216. The non-transitory computer-readable medium of claim 215, wherein a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

217. 211. The non-transitory computer readable medium of any one of claims 188-210, wherein the prediction of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

218. 211. The non-transitory computer readable medium of any one of claims 188-210, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

219. 1. A non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: below: one or more neuroaxonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immune-modulating biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; or one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN. obtaining or having obtained a dataset comprising expression levels of a plurality of biomarkers including at least one of: generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; An order to perform The non-transitory computer-readable medium comprising:

220. 1. A non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: below: one or more neuroaxonal integrity biomarkers selected from the group consisting of CNTN2, FLRT2, NEFL, PRTG, SERPINA9, OPG, GFAP, and TNFRSF10A; one or more neuroinflammatory biomarkers selected from the group consisting of CCL20, GH, TNFRSF10A, CXCL9, CXCL13, IL-12B, CD6, and TNFSF13B; one or more immune-modulating biomarkers selected from the group consisting of CDCP1, CD6, CXCL9, CXCL13, IL-12B, and TNFSF13B; one or more myelination biomarkers selected from the group consisting of MOG, APLP1, and OPN; or one or more cerebrovascular function biomarkers selected from the group consisting of COL4A1, VCAN, GFAP, and CD6 obtaining a dataset comprising expression levels of a plurality of biomarkers, including at least one of: generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; An order to perform The non-transitory computer-readable medium comprising:

221. 221. The non-transitory computer-readable medium of claim 219 or 220, wherein the one or more axonal integrity biomarkers include NEFL, OPG, and GFAP, the one or more neuroinflammation biomarkers include CXCL13 and CXCL9, the one or more immune modulation biomarkers include CDCP1, and the one or more myelination biomarkers include MOG and APLP1.

222. 222. The non-transitory computer-readable medium of claim 221, wherein the one or more axonal integrity biomarkers further comprise SERPINA9, FLRT2, and CNTN2; the one or more neuroinflammation biomarkers further comprise CCL20, CXCL9, TNFRSF10A, and CD6; the one or more immune modulation biomarkers further comprise TNFSF13B; and the one or more myelination biomarkers further comprise OPN.

223. 223. The non-transitory computer readable medium of claim 222, wherein the one or more neuroaxonal integrity biomarkers further comprise PRTG and the one or more immune modulatory biomarkers further comprise IL-12B.

224. The performance of the predictive model has a correlation coefficient (R ２ 224. The non-transitory computer-readable medium of claim 223, characterized by:

225. 224. The non-transitory computer-readable medium of claim 223, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

226. 224. The non-transitory computer-readable medium of claim 223, wherein the performance of the predictive model is characterized by a PPV of at least 0.

17.

227. 220. The non-transitory computer-readable medium of claim 219, wherein the plurality of biomarkers comprises one or more axon integrity biomarkers, and wherein the one or more axon integrity biomarkers comprises GFAP.

228. 228. The non-transitory computer-readable medium of claim 227, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

229. 229. The non-transitory computer-readable medium of claim 227 or 228, wherein the performance of the predictive model is characterized by an AUROC between 0.72 and 0.

78.

230. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 228. The non-transitory computer-readable medium of claim 227, characterized by a coefficient.

231. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ 231. The non-transitory computer-readable medium of claim 227 or 230, characterized by a coefficient.

232. The non-transitory computer-readable medium of claim 219, wherein the plurality of biomarkers does not include GFAP.

233. 233. The non-transitory computer-readable medium of claim 232, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

234. 234. The non-transitory computer-readable medium of claim 232 or 233, wherein the performance of the predictive model is characterized by an AUROC of between 0.66 and 0.

70.

235. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 233. The non-transitory computer-readable medium of claim 232, characterized by a coefficient.

236. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 236. The non-transitory computer-readable medium of claim 232 or 235, characterized by a coefficient.

237. 237. The non-transitory computer readable medium of any one of claims 219 to 236, wherein the prediction of multiple sclerosis disease progression is a brain parenchymal fraction value.

238. 237. The non-transitory computer readable medium of any one of claims 219 to 236, wherein the prediction of multiple sclerosis disease progression is an Expanded Disability Status Scale (EDSS) score.

239. The non-transitory computer-readable medium of claim 238, wherein an Expanded Disability Status Scale (EDSS) score of less than 6 indicates mild / moderate MS disease progression, and an EDSS score of 6.0 or greater indicates severe MS disease progression.

240. 237. The non-transitory computer readable medium of any one of claims 219-236, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score.

241. The non-transitory computer readable medium of claim 240, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression.

242. The non-transitory computer-readable medium of claim 241, wherein a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

243. 237. The non-transitory computer readable medium of claims 219-236, wherein the prediction of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

244. 237. The non-transitory computer readable medium of claims 219-236, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

245. 1. A non-transitory computer readable medium for predicting multiple sclerosis disease progression in a subject, the non-transitory computer readable medium comprising: When executed by a processor, the processor: obtaining a dataset comprising expression levels of a plurality of biomarkers, the plurality of biomarkers comprising two or more of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-α, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG; generating a prediction of multiple sclerosis disease progression by applying a predictive model to the expression levels of the plurality of biomarkers; An order to perform The non-transitory computer-readable medium comprising:

246. 246. The non-transitory computer readable medium of claim 245, wherein the plurality of biomarkers comprises each of GFAP, CDCP1, MOG, CXCL13, OPG, APLP1, VCAN, NEFL, CXCL9, TNFRSF10A, CCL20 / MIP 3-alpha, TNFSF13B, CD6, SERPINA9, FLRT2, OPN, CNTN2, COL4A1, GH, IL-12B, and PRTG.

247. The performance of the predictive model has a correlation coefficient (R ２ 247. The non-transitory computer-readable medium of claim 246, characterized by:

248. 248. The non-transitory computer-readable medium of claim 247, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

74.

249. 248. The non-transitory computer-readable medium of claim 247, wherein the performance of the predictive model is characterized by a PPV of at least 0.

17.

250. The non-transitory computer-readable medium of claim 245, wherein the plurality of biomarkers comprises GFAP.

251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises CDCP1.

252. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises APLP1.

253. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises CXCL13.

254. 251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises MOG.

255. 251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises OPG.

256. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1 and APLP1.

257. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises MOG and CDCP1.

258. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises APLP1 and CXCL13.

259. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1 and SERPINA9.

260. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises MOG and CXCL13.

261. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1, CCL20, and APLP1.

262. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1, APLP1, and CXCL13.

263. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1, CCL20, and MOG.

264. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1, APLP1, and SERPINA9.

265. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CDCP1, MOG, and APLP1.

266. 266. The non-transitory computer-readable medium of any one of claims 250-265, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

70.

267. 267. The non-transitory computer-readable medium of any one of claims 250-266, wherein the performance of the predictive model is characterized by an AUROC between 0.72 and 0.

78.

268. 251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises MOG.

269. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises APLP1.

270. 251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises OPG.

271. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises TNFRSF10A.

272. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises CDCP1.

273. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises APLP1.

274. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises NEFL.

275. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises CNTN2.

276. 251. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises GH.

277. The non-transitory computer readable medium of claim 250, wherein said plurality of biomarkers further comprises CXCL9.

278. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises OPG and MOG.

279. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises OPG and APLP1.

280. The non-transitory computer-readable medium of claim 250, wherein the plurality of biomarkers further comprises TNFRSF10A and MOG.

281. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL9 and OPG.

282. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises TNFRSF10A and APLP1.

283. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises APLP1 and NEFL.

284. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL13 and APLP1.

285. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises FLRT2 and APLP1.

286. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL9 and APLP1.

287. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises GH, and APLP1.

288. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL9, OPG, and MOG.

289. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CNTN2, OPG, and MOG.

290. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL9, OPG, and APLP1.

291. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises OPG, RPGG, and MOG.

292. 251. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises OPG, OPN, and MOG.

293. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL13, APLP1, and NEFL.

294. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises FLRT2, APLP1, and NEFL.

295. The non-transitory computer-readable medium of claim 250, wherein the plurality of biomarkers further comprises OPN, APLP1, and NEFL.

296. 251. The non-transitory computer-readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL9, APLP1, and NEFL.

297. The non-transitory computer readable medium of claim 250, wherein the plurality of biomarkers further comprises CXCL13, FLRT2, and APLP1.

298. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 300. The non-transitory computer-readable medium of any one of claims 268 to 297, characterized by a coefficient.

299. The performance of the predictive model is between 0.11 and 0.22, with a Pearson R ２ 300. The non-transitory computer-readable medium of any one of claims 268 to 298, characterized by a coefficient.

300. The non-transitory computer-readable medium of claim 245, wherein said plurality of biomarkers does not include GFAP.

301. The non-transitory computer readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1 and OPG.

302. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1 and SERPINA9.

303. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and TNFRSF10A.

304. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and MOG.

305. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1 and MOG.

306. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, MOG, and OPG.

307. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, SERPINA9, and OPG.

308. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, OPG, and CXCL13.

309. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, CXCL9, and OPG.

310. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, FLRT2, and OPG.

311. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, MOG, OPG, and CXCL13.

312. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, MOG, TNFRSF10A, and OPG.

313. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, CXCL9, SERPINA9, and OPG.

314. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, CNTN2, SERPINA9, and OPG.

315. The non-transitory computer readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, SERPINA9, CD6, and OPG.

316. 316. The non-transitory computer-readable medium of any one of claims 300-315, wherein the performance of the predictive model is characterized by an AUROC of at least 0.

65.

317. 317. The non-transitory computer-readable medium of any one of claims 300-316, wherein the performance of the predictive model is characterized by an AUROC of between 0.66 and 0.

70.

318. The non-transitory computer readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and NEFL.

319. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and OPN.

320. The non-transitory computer readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and FLRT2.

321. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG and MOG.

322. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL9 and OPG.

323. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises GH and NEFL.

324. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL13 and NEFL.

325. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises APLP1 and NEFL.

326. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CCL20 and NEFL.

327. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL9 and NEFL.

328. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG, MOG, and NEFL.

329. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG, FLRT2, and NEFL.

330. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL9, OPG, and NEFL.

331. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG, CDCP1, and NEFL.

332. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises OPG, OPN, and NEFL.

333. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises GH, APLP1, and NEFL.

334. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises GH, CXCL13, and NEFL.

335. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises GH, CDCP1, and NEFL.

336. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL13, CCL20, and NEFL.

337. The non-transitory computer readable medium of claim 300, wherein the plurality of biomarkers comprises GH, CCL20, and NEFL.

338. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL.

339. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and NEFL.

340. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL.

341. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL.

342. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and MOG.

343. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CDCP1, CXCL13, MOG, and NEFL.

344. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and NEFL.

345. 301. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CXCL9, CXCL13, MOG, and NEFL.

346. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CXCL9, CXCL13, and NEFL.

347. The non-transitory computer-readable medium of claim 300, wherein the plurality of biomarkers comprises CD6, CDCP1, CXCL13, and MOG.

348. The performance of the predictive model is at least Pearson's R of 0.

10. ２ 348. The non-transitory computer-readable medium of claims 318-347, characterized by a coefficient.

349. The performance of the predictive model is such that the Pearson R is between 0.015 and 0.

090. ２ 349. The non-transitory computer-readable medium of claims 318-348, characterized by a coefficient.

350. 350. The non-transitory computer readable medium of any one of claims 245-349, wherein the prediction of multiple sclerosis disease progression is a brain parenchymal fraction value.

351. 350. The non-transitory computer readable medium of any one of claims 245-349, wherein the prediction of multiple sclerosis disease progression is Expanded Disability Status Scale (EDSS) score.

352. The non-transitory computer-readable medium of claim 351, wherein an Expanded Disability Status Scale (EDSS) score of 6 or less indicates mild / moderate MS disease progression and an EDSS score of greater than 6.5 indicates severe MS disease progression.

353. 350. The non-transitory computer readable medium of any one of claims 245-349, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score.

354. The non-transitory computer readable medium of claim 353, wherein the prediction of multiple sclerosis disease progression is a Patient-Determined Disease Stage (PDDS) score that distinguishes between severe MS disease progression and mild / moderate MS disease progression.

355. The non-transitory computer-readable medium of claim 354, wherein a Patient-Determined Disease Stage (PDDS) score of 4 or less indicates mild / moderate MS disease progression, and a PDDS score of greater than 4 indicates severe MS disease progression.

356. 350. The non-transitory computer readable medium of any one of claims 245-349, wherein the prediction of multiple sclerosis disease progression is a PRO Measurement Information System (PROMIS) score.

357. 350. The non-transitory computer readable medium of any one of claims 245-349, wherein the prediction of multiple sclerosis disease progression is the Multiple Sclerosis Rating Scale-Revised (MSRS-R) score.

358. The non-transitory computer-readable medium of any one of claims 188 to 357, wherein generating a prediction of multiple sclerosis disease progression by applying the predictive model to expression levels of a plurality of biomarkers further comprises applying the predictive model to one or more subject attributes of the subject, wherein the subject attributes include any of age, sex, and disease duration.

359. The non-transitory computer-readable medium of any one of claims 188-358, wherein generating the prediction of multiple sclerosis disease progression comprises comparing a score output by the predictive model to a reference score.

360. The reference score is A) EDSS score; B) Brain parenchymal fraction value; C) PDDS score; D) PROMIS score; or E) MSRS-R score 360. The non-transitory computer-readable medium of claim 359, corresponding to any one of:

361. The non-transitory computer-readable medium of claim 360, wherein the reference score further corresponds to mild / moderate MS disease progression or severe MS disease progression.

362. 362. The non-transitory computer readable medium of any one of claims 188-361, wherein the expression levels of the plurality of biomarkers are determined from a test sample obtained from the subject.

363. The non-transitory computer-readable medium of claim 362, wherein the test sample is a blood or serum sample.

364. The non-transitory computer readable medium of claim 362 or 363, wherein the subject has, is suspected of having, or has previously been diagnosed with multiple sclerosis.

365. 365. The non-transitory computer readable medium of any one of claims 188-364, wherein obtaining or having obtained the dataset comprises performing an immunoassay to determine the expression levels of the plurality of biomarkers.

366. The non-transitory computer-readable medium of claim 365, wherein the immunoassay is a proximity extension assay (PEA) or a LUMINEX xMAP multiplex assay.

367. 367. The non-transitory computer-readable medium of claim 365 or 366, wherein performing the immunoassay comprises contacting the test sample with a plurality of reagents including antibodies.

368. The non-transitory computer-readable medium of claim 367, wherein the antibody comprises one of a monoclonal antibody and a polyclonal antibody.

369. The non-transitory computer-readable medium of claim 367, wherein the antibodies include both monoclonal and polyclonal antibodies.

370. instructions that, when executed by the processor, cause the processor to select a therapy for administration to the subject based on the prediction of multiple sclerosis disease progression.

370. The non-transitory computer-readable storage medium of any one of claims 188 to 369, further comprising:

371. instructions that, when executed by the processor, cause the processor to determine the therapeutic effectiveness of a therapy previously administered to the subject based on the prediction of multiple sclerosis disease progression.

371. The non-transitory computer-readable storage medium of any one of claims 188 to 370, further comprising:

372. instructions for causing the processor to determine therapeutic efficacy of the therapy, instructions that, when executed by the processor, cause the processor to compare the prediction with a previous prediction determined for the subject at a previous time.

372. The non-transitory computer-readable storage medium of claim 371, further comprising:

373. instructions for causing the processor to determine therapeutic efficacy of the therapy, instructions that, when executed by the processor, cause the processor to determine that the treatment is indicative of efficacy according to a difference between the prediction and a previous prediction.

373. The non-transitory computer-readable storage medium of claim 372, further comprising:

374. instructions for causing the processor to determine therapeutic efficacy of the therapy, instructions that, when executed by the processor, cause the processor to determine that the treatment is lacking in efficacy in response to a lack of difference between the prediction and the previous prediction.

373. The non-transitory computer-readable medium of claim 372, further comprising:

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