Journal of the Neurological Sciences 305 (2011) 110

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Journal of the Neurological Sciences

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Biomarkers of disease activity in multiple sclerosis

Jerome J. Graber a, Suhayl Dhib-Jalbut b,
a b

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Department of Neurology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

a r t i c l e

i n f o

a b s t r a c t
As therapeutic options for multiple sclerosis widen, validated biomarkers of clinical disease activity are urgently needed. Reliable biomarkers would assist in choosing initial therapy, monitoring response to therapy, detecting subclinical disease activity, predicting and possibly preventing therapeutic failure, and hopefully improving both short (relapses) and long-term (disability) outcomes. The presence of oligoclonal bands in the cerebrospinal uid is a well-validated biomarker that is useful in initial diagnosis. Neutralizing antibodies to interferon-beta are also useful in identifying treatment failure and possibly guiding changes in therapy. The discovery of antibodies to aquaporin-4 in patients with neuromyelitis optica delineates patients with a fundamentally different underlying pathophysiology and clinical course who may require alternate therapeutic approaches. While numerous other candidate biomarkers in serum and cerebrospinal uid have been described, none so far have the validated reliability necessary for widespread clinical use. The availability of multiple genetic and protein microarray technology may assist in identifying more reliable candidate biomarkers or patterns of multiple biomarkers and improve specicity. The heterogeneity of multiple sclerosis may necessitate individualized biomarkers and therapeutic decisions within distinct subsets of patients. 2011 Elsevier B.V. All rights reserved.

Article history: Received 5 January 2011 Received in revised form 25 February 2011 Accepted 1 March 2011 Available online 3 April 2011 Keywords: Multiple sclerosis Biomarker Disease activity Interferon Glatiramer Interleukin Cytokine T cell

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . 2. MS immunopathogenesis . . . . . . . . . . . . . 3. Cytokines . . . . . . . . . . . . . . . . . . . . 4. T cell subsets . . . . . . . . . . . . . . . . . . 5. B cells and immunoglobulins . . . . . . . . . . . 6. Natural killer cells . . . . . . . . . . . . . . . . 7. Dendritic cells and macrophages . . . . . . . . . 8. Adhesion molecules and matrix metalloproteinases 9. Chemokines . . . . . . . . . . . . . . . . . . . 10. Other markers . . . . . . . . . . . . . . . . . . 11. CSF biomarkers . . . . . . . . . . . . . . . . . 12. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2 4 4 5 5 5 6 6 6 7 8

1. Introduction Multiple sclerosis (MS) is one of the most common causes of neurologic disability in young adults with an enormous impact on quality of life and societal costs due to lost work and cost of providing
Corresponding author at: Department of Neurology, UMDNJ-Robert Wood Johnson Medical School, 97 Paterson Street, Room 205, New Brunswick, NJ 08901, United States. Tel.: + 1 732 235 7732; fax: + 1 732 235 8115. E-mail address: jalbutsu@umdnj.edu (S. Dhib-Jalbut). 0022-510X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.03.026

care. An expanding array of treatment options with different mechanisms of action have been shown to reduce relapse rates, MRI activity and accumulation of permanent disability [1]. However, continued relapses and eventual disability are still expected in the majority of patients on available therapies. Little denitive data exists to guide clinicians which treatment option is most effective for any particular patient, or when relapses or MRI activity indicate expected breakthrough disease activity or true treatment failure. As more targeted therapies become available, rational treatment using biomarkers of disease activity may become useful in predicting responsiveness to

J.J. Graber, S. Dhib-Jalbut / Journal of the Neurological Sciences 305 (2011) 110 Table 2 Challenges to biomarker discovery and validation.

different treatment options and guiding when changes in therapy are warranted to prevent relapses and accumulation of permanent disability (Table 1). Biomarkers may also be useful in characterizing different disease states, for example, proteomics analysis has found different protein expression in primary progressive compared to relapsing remitting MS [2]. Reliable biomarkers that help guide treatment decisions to prevent eventual disability have the potential to vastly improve outcomes and cost-effective care. Several obstacles exist in developing biomarkers useful for clinical decisions (Table 2). One major difculty is that there is no consensus of what denes active disease, since patients without clinical evidence of relapse may have changes on MRI suggesting subclinical disease activity and patients with progressive forms of disease continue to decline in function without obvious relapses or MRI changes. MRI activity modestly predicts long term disease outcomes, depending on which measures are used (enhancing lesions, T2 lesions, T1 black holes, white matter atrophy, gray matter atrophy, etc.) [3]. However, MRI studies have also shown that a substantial number of brain inammatory events are subclinical without active clinical changes [3]. The relevance of subclinical MRI changes in the absence of clinical relapse is not rmly established, resulting in the adage treat the patient, not the scan, though much hand-wringing and anxiety on the part of patient and physician can result. Some relapses may be marked by non-traditional clinical decits such as psychiatric and cognitive changes, which nonetheless signicantly impact patients' lives. Patients may suffer exacerbations of prior decits due to impaired conductance across partially remyelinated axons in the absence of CNS inammation due to active infection or medications that slow axonal conductance, mimicking clinical relapse (pseudo-relapse). Disease activity also substantially varies both between individuals and within individual patients over time, so that brief periods of frequent relapse and MRI changes may be followed by disease quiescence, creating the problem of false positive ndings via regression to the mean in clinical trials that lack direct comparative arms. Determining when such episodes portend treatment failure in individual patients is a major challenge to the clinician. Studies of biomarkers are also hampered by signicant normal variability in immunological variables, seasonally and even diurnally [4]. In addition, many serum biomarkers are non-specic indicators of systemic inammation and may also be altered during even minor infections [4]. Another obstacle to interpreting studies of biomarkers in response to therapy is differences in the formulations of IFN- used in different studies, which have been shown to have different effects on biomarkers of disease [57]. Numerous biomarkers of disease activity have been explored encompassing cytokines, chemokines, immune cell subsets, costimulatory molecules, antibodies, vascular adhesion and permeability and neuronal and glial damage, among others (Tables 38). 2. MS immunopathogenesis Studies of MS patients highlight that numerous aspects of their immune systems are dysfunctional (Fig. 1) [8]. Much attention has

Highly variable clinical course Heterogeneous mechanisms of disease Nonspecicity of disease markers Intra- and inter-individual variability in markers Variable responses to treatment Diagnosis of exclusion without gold standard (numerous mimics) Frequent subclinical disease activity Modest correlation of MRI features and relapse rates with long term outcomes (xed disability) Heterogeneous treatments with various mechanisms of action highlighted overactivity and invasion of Th1 and Th17 cells that secrete proinammatory cytokines, as well as CD8+ effector cells [8]. There also appears to be loss of activity of regulatory T cells that normally keep inammation in check [8,9]. In addition, evidence supports abnormal activity of B cells, secreted antibodies and complement activation [8]. Recent evidence also supports that dendritic cells, macrophages and natural killer cells are also dysfunctional [8,10]. Molecules that regulate the integrity of the blood brain barrier and invasion of inammatory cells into the normally protected CNS also appear to be abnormal in patients with MS [8]. The underlying immunopathogenesis of MS may differ between patients and change over the course of each patient's lifespan, creating further difculties in identifying and characterizing individual biomarkers of disease that apply to all patients. 3. Cytokines Changes in cytokine secretion by immune cells mark activity and function and affect surrounding immune cells, vasculature and tissue. Because of their known role in MS pathogenesis, numerous cytokines have been explored as biomarkers (Table 3). T cell cytokine activity is generally divided into four subsets: Th1 (proinammatory activity with IFN-, IL-12 and TNF- secretion), Th2 (immunoregulatory activity with IL-10 and IL-4 secretion), Th17 (inammatory IL-17 and IL-6 secretion) and Treg (variably FoxP3+ or CD4+CD25high immunoregulatory cells). This simplistic view is hampered by the coexistence of other subsets of cells in both CD4+ and CD8+ populations and their variable responsiveness and survival in different environments as well as their complex interactions with other cells. Administration of IFN- to patients with MS appeared to trigger acute exacerbations, and serum levels of IFN- production increase prior to relapse, supporting the idea that MS activity is primarily associated with Th1 activity [1113]. IFN- decreases peripheral blood mononuclear cell (PBMC) production of IFN-, particularly in patients who demonstrate a clinical response to treatment [7]. Patients who have clinical response to glatiramer

Table 1 Potential importance of biomarkers in MS.

Table 3 Potential serum biomarkers of disease activity in multiple sclerosis. Biomarker Cytokines TNF- (serum and CSF) IL-10 (serum and CSF) IL-12 (serum and CSF) IL-17 (serum and CSF) IFN- Osteopontin (CSF) IL-6 (CSF) IFN = interferon. IL = interleukin. TNF = tumor necrosis factor. Correlate Relapse (+), Disability (+) Relapse (), Treatment Response Relapse (+) Relapse (+), Treatment Response Relapse (+), Treatment Response Disease activity (+) NMO severity (+) Reference [70,71] [7,18,31] [1518] [23,25,26] [7,12,14] [114] [115,144]

Accurate and earlier diagnosis (e.g. CIS, RRMS, not MS) Prediction of short term risk of clinical disease activity (relapse, MRI activity, conversion to RRMS) Assessment of ongoing subclinical disease activity Prediction of long term clinical disease course (conversion to SPMS, accumulation of xed disability) Surrogate outcome for clinical trials Correlation with various pathological subtypes Prediction of response to different treatment options Assessment of treatment failure/resistance

J.J. Graber, S. Dhib-Jalbut / Journal of the Neurological Sciences 305 (2011) 110 Table 4 Cell surface biomarkers. Biomarker Cell Surface Markers K2P5.1 + T cells IFN- Receptor- IFN- Receptor-2 CD56bright NK cells CD8 + CD25 + FoxP3 + Treg cells Fas/FasL CD80 CD86 CD40 PD1/PDL1 PDL2 Survivin IFN = interferon. Ig = immunoglobulin. IL = interleukin. NK = natural killer. PD = programmed cell death. PDL = programmed cell death ligand. Correlate Relapse Relapse, Treatment Response Treatment Response Relapse Relapse Relapse, Disability Relapse Relapse, Treatment Response Treatment Response Relapse Treatment Respone Relapse, Treatment Response Reference [45] [33] [34] [10,68] [35] [41,42] [3638] [3639] [39] [40] [39] [43] Table 6 Biomarkers related to adhesion and migration. Biomarker Adhesion and migration sVCAM (serum and CSF) LFA1 VLA4 Correlate Reference [7,76,117] [77] [76] [19,79,81] [80] [19] [86] [82] [82] [32,82] [83,120] [87,88] [89,118,119] [90] [91] [116,117] [118] [124]

Relapse (), treatment response Relapse (+) Relapse (+), treatment response, disability (+) MMP9 Relapse (+), treatment response TIMP1 Relapse () MMP-8 Relapse (+) IL-8 Relapse IP-10 (serum and CSF) Relapse (+) CXCL8 Relapse (+) CCL2 (serum and CSF) Relapse () CCL5 (serum and CSF) Relapse (+) CXCR3 Relapse (+) CXCL13 (serum and CSF) Disease activity (+), treatment response CCR5 Relapse CX3CR1 Relapse () ICAM (CSF) Relapse (+) CXCL12 (CSF) Relapse NCAM (CSF) Relapse

acetate also show reduction of PBMC IFN- expression compared to patients who do not respond [14]. Later it was discovered that periods of systemic IL-12 production (which stimulates IFN-, Th1, Th17 and NK cell subsets) precede MS relapse and correlate with clinical and MRI disease activity [1519]. IL-12 levels in serum also decrease after treatment with glatiramer acetate [20]. IL-12 is a heterodimer composed of p40 and p35 components, and it was subsequently found that the IL-12p40 subunit can also complex with p19 to form IL23, which promotes a unique subset of IL-17 producing Th17 cells depending on the cytokine milieu present [21]. Baseline levels of IL12p35 mRNA in blood predicted with 80% accuracy decreased clinical activity after treatment with IFN- in one study [22]. IL-17 production is increased in relation to disease activity, and decreased by IFN- therapy [23,24]. Levels of PBMC IL-17 production are higher in patients recently diagnosed with MS compared to those with longstanding disease, which may relate to recent or severe disease activity [25]. Nonresponders to IFN- treatment have higher levels of serum IL-17F protein, suggesting heterogeneity of immunopathogenesis in individual MS patients with implications for treatment [26]. While the data that IL-12 and IL-23/17 are associated with disease activity in MS is overwhelming, a randomized trial of patients treated with antibody

CSF = cerebrospinal uid. ICAM = intercellular adhesion molecule. IL = interleukin. LFA = leukocyte function-associated antigen. MMP = matrix metalloprotease. NCAM = neural cell adhesion molecule. TIMP = tissue inhibitor of metalloprotease. VCAM = vascular cell adhesion molecule. VLA = very late antigen.

against the p40 subunit of both IL-12 and IL-23 failed to achieve any benet on MRI or clinical disease activity [27]. IL-10 is an immunoregulatory cytokine produced by Th2 cells. Systemic IL-10 decreases prior to clinical and MRI relapse, and increases when disease activity resolves [18,28,29]. Treatment with IFN- or glatiramer acetate augments systemic IL-10 activity [30,31]. Lower baseline serum levels of IL-10 predict clinical response to IFN- [32]. Patients who continue to have clinical relapses and MRI activity after IFN- treatment had a paradoxical decrease in serum IL-10 levels [7].

Table 7 Biomarkers of tissue damage and repair. Biomarker Correlate Relapse (+), conversion to RRMS, disability Disability (+) Relapse (+) Disease activity (+) Relapse (+) Disability Reference [128,130] [126] [125] [131] [121,122] [102]

Table 5 Potential humoral and antibody biomarkers. Biomarker Humoral and Antibody OCB (CSF) Aquaporin-4 (NMO) antibody IFN- Neutralizing Antibodies CD19 + CD138 + B cells (serum and CSF) MOG/MBP antibodies CD46/59 antibodies Complement factor H C4 fragment EBNA IgG Ig kappa light chains (CSF) Correlate Conversion to RRMS (+), relapse (+), disability (IgM) NMO disease type (+) Relapse (+), treatment response Relapse (+) Relapse (+), conversion to RRMS Relapse (+) Relapse (+), disability Disease activity (+) Disease activity (+) Relapse (+) Reference [105,106] [46] [52] [55] [56] [64] [65] [66] [60] [76]

Markers of tissue damage Neurolament chains (CSF) GFAP (CSF) S100 (CSF) NAA (CSF) Nitric oxide products (CSF) Pentosidine Growth factors BDNF CNTF (CSF) GDNF NGF NT3 NT4

Relapse recovery response Relapse Relapse recovery Relapse recovery Relapse recovery Relapse recovery

(+), treatment

[9799] [124] [99] [99] [99] [99]

(+) (+) (+) (+)

C = complement. Ig = immunoglobulin. MOG = myelin oligodendrocyte glycoprotein. MBP = myelin basic protein. NMO = neuromyelitis optica. OCB = oligoclonal bands. RRMS = relapsing remitting multiple sclerosis.

CNTF = ciliary neurotrophic factor. CSF = cerebrospinal uid. Ig = immunoglobulin. GDNF = Glial Derived Neurotrophic Factor. GFAP = glial brillary acidic protein. NGF = Neural Growth Factor. NT = Neurotrophin. RRMS = relapsing remitting multiple sclerosis.

4 Table 8 Other potential biomarkers. Biomarker Other Myxovirus resistance protein A Myoinositol Bri2-23 (CSF) Fetuin-A (CSF) ILT3 Correlate

J.J. Graber, S. Dhib-Jalbut / Journal of the Neurological Sciences 305 (2011) 110

Reference [9395] [137] [134] [134] [63]

Relapse (), treatment response (+) Cognitive impairment Disability (+) Active SPMS (+) Relapse ()

ILT = immunoglobulin-like transcript.

Multiple studies have also found that the ratio of IL-10/IL-12 correlates with disease activity, and improves with IFN- treatment in patients who respond to therapy [7]. 4. T cell subsets Cell surface markers help to dene various cell types and their state of activity in MS (Table 4). Various T cell subsets expressing different cytokine populations (see above) have long been known to play a role in MS disease activity. Newer T cell populations are being described that may also inuence MS pathophysiology. Expression of IFN- receptor beta (IFNR) increases susceptibility to apoptosis in activated T cells, and low levels of IFNR expression are found in active MS and improve with IFN- therapy [33]. IFN- treatment produces downregulation of IFN- receptors by internalization, and pre-treatment IFN- receptor-2 levels may correlate with later IFN- responsiveness, because individuals with low pre-treatment IFN-

receptor 2 levels are signicantly more likely to develop neutralizing antibodies [34]. A population of FoxP3 expressing CD8+ T cells exerting immunoregulatory effects on activated T cells and dendritic cells has been described. The number and activity of those cells are reduced in serum and CSF during acute MS relapse [35]. Levels of the Th1 stimulatory molecule CD80 increase during active MS, while expression of the immunoregulatory CD86 molecule decline; IFN- treatment appears to reverse these changes which correlate with clinical response [3638]. Surface levels of CD40 are also stimulated by IFN- treatment and correlate with relapse rates and stable EDSS [39]. The costimulatory pair of PD1/PDL1 increases in inactive compared to active disease and correlates with increased IL-10 production [40]. PDL2 expression on monocytes is stimulated by IFN- treatment and higher induction is associated with clinical response [39]. Higher levels of the cell surface ligands Fas and FasL which promote apoptosis of activated T cells predict slower long-term disability progression in MS [41]. During acute relapse, serum FasL declines while Fas increases [42]. Survivin, a molecule that inhibits apoptosis of activated T cells, was found to be increased during active disease and down-regulated by IFN- [43,44]. Recent studies have identied a variety of potassium channels that are important in activation of different immune cell subsets. In particular, the potassium channel K2P5.1 has been found to be upregulated in T cells of MS patients during acute relapse [45]. 5. B cells and immunoglobulins Though T cells clearly play a role in MS, there has been growing appreciation of the role of the humoral immune system and antibodies

Fig. 1. Theorized factors in the pathogenesis of multiple sclerosis.

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in MS (Table 5). Identication of the neuromyelitis optica (NMO) antibody to aquaporin-4 predicts a different clinical course of aggressive disease with predominant spinal cord and optic nerve disease and likely accumulation of early xed disability as well as death compared to traditional multiple sclerosis [46]. The clinical differences are so striking that some experts consider NMO a completely separate disease from MS, though cases with overlapping features do occur and a wider spectrum of clinical NMO is now being described besides the traditional concept of bilateral optic neuritis with simultaneous longitudinal transverse myelitis [46]. As many as 2040% of patients meeting clinical criteria for diagnosis of NMO will not have detectable serum antibody, depending on the method used [46,47]. The optimal treatment of NMO is controversial, but NMO patients with antibodies to aquaporin-4 may have severe exacerbation of disease after IFN- treatment, suggesting that the aquaporin-4 antibody may also predict treatment response [48,49]. Limited data exists, but proposed treatments differ from those for traditional MS and include plasmapheresis, B cell depletion with antibody against CD20 (rituximab) and long term immunosuppression with corticosteroids, immunoglobulin preparations, azathioprine or mitoxantrone [46]. Recent reports have identied an individual who had detectable NMO antibody years prior to any clinical disease, suggesting antibody may exist long prior to disease activity [50]. CSF positive for aquaporin4 in the absence of serum NMO has also been reported, though larger investigations suggest this may be a rare event [47,51]. There is now overwhelming evidence that patients who develop neutralizing antibodies to IFN- are more likely to have increased radiological and clinical disease activity [reviewed in 52]. An expert panel produced clinical guidelines recommending periodic monitoring of neutralizing antibodies in patients on IFN- therapy and use of neutralizing antibody titers in clinical decisions in the context of the clinical status of the patient [52]. There is also evidence to suggest that patients with neutralizing antibodies to natalizumab may have recurrent disease activity [53]. The signicance of glatiramer-binding antibodies is unclear, but some studies have suggested that low-titer glatiramer antibodies may be associated with better outcomes [53,54]. The presence of mature B cell populations (as measured by CD19+ CD138+ cells) correlate with active disease [55]. Patients with serum antibodies to MOG and MBP may have increased relapse rates and higher conversion from CIS to RRMS, compared to patients without such antibodies, though others have not found such an association [5658]. Other studies on the inuence of antibodies to various myelin antigens have provided conicting results, however antibodies against galactocerebroside have been reported to be specic for RR and SPMS but not found in healthy controls and only rarely present in CIS and PPMS [59]. Serum levels of antibodies against Epstein Barr Nuclear Antigen (EBNA) have been reported to correlate with MRI measures of disease activity, but others have failed to conrm this nding [60,61]. Disparities between these ndings may reect differences in denitions of disease activity (in terms of clinical relapses or MRI activity). Antibodies to heat shock proteins (especially HSP 70) may differentiate between forms of MS, but a clear relationship with disease activity has not been conrmed yet [62]. A group of immunoglobulin-like transcripts on the surface of monocytes appear to exert regulatory effects on activated T cells. One of these, immunoglobulin-like transcript-3 (ILT3), a cell surface protein regulating immune stimulation, has been found to be downregulated during MS relapse, theoretically decreasing regulatory effects and restored during remission and IFN- treatment [63]. One study found antibodies to the complement regulators CD46 and CD59 during active but not stable disease [64]. Serum levels of complement regulatory factor H also are transiently increased during acute relapse and progressive forms of MS, and may predict development of relapse and progression [65]. Various degradation fragments of C4 serve as markers of antibody binding to target antigens and subsequent

inammation and a particular fragment of complement C4 correlates with disease activity in MS [66]. 6. Natural killer cells Recent trials of daclizumab, an antibody blocking the IL-2 receptor (CD25), have brought to light the possible role of natural killer (NK) cells in CNS autoimmunity [67]. While the majority of NK cells mediate cytotoxicity against MHC-1 decient targets, a minority (10%) population of CD56bright cells appears to play a targeted immunoregulatory role by granzyme and perforin-dependent killing of activated T cells and immature dendritic cells [67]. Clinical and MRI periods of disease activity are associated with declines in the NK cell population, though whether this represents cause, effect, or a sequestration phenomenon wherein these cells home in to the CNS and are less apparent in systemic blood is unknown [10,68]. Daclizumab therapy augments this CD56bright regulatory NK cell population which then act to suppress activated T cells, with promising clinical results [67,69]. 7. Dendritic cells and macrophages Cytokines primarily derived from cells of the innate immune system (macrophages, microglia and dendritic cells) also have been found to play a signicant role in MS. TNF- is a prominent inammatory cytokine produced by cells of the innate immune system as well as T cells. TNF- levels are increased in MS patients compared to healthy controls and higher levels correlate with risk of disability progression [70,71]. However, treatments inducing TNF- blockade through various mechanisms induced exacerbations in clinical trials and new onset autoimmune demyelination in patients with rheumatoid arthritis [72,73]. Inammatory osteopontin levels primarily produced by activated macrophages correlate with disease activity [74]. MicroRNAs (miRNAs) are short sequences of RNA that act by binding to complementary mRNA sequences resulting in their degradation or suppression of translation into protein. Several microRNAs important in expression of CD47, a cell surface molecule that protects from macrophage phagocytosis, have been found to be differentially expressed in active MS brain lesions [75]. 8. Adhesion molecules and matrix metalloproteinases Adhesion molecules and their receptors help activated inammatory cells enter target organs, including the CNS. Several adhesion molecules and receptors have been found to play roles in inltration of inammatory cells into the CNS in MS (Table 6). Activated T (and B) cells leave circulation and enter target organs by the expression of surface molecules (very late antigen-4 (VLA-4), LFA-1) which bind receptors (VCAM and ICAM) on stimulated endothelial cells. Levels of soluble vascular cell adhesion molecule (sVCAM) increase with IFN- treatment in a dose-dependent fashion and correlate with reductions in MRI activity and long term disability progression [6,76]. Soluble VCAM may function to reduce adhesion of peripheral activated T cells by binding cells in the circulation and blocking their binding to membrane-bound VCAM on endothelial cells [6]. Downregulation of VLA-4 on T cells after IFN- therapy also correlates with slower progression of EDSS, supporting the importance of this pathway [74]. LFA-1, the adhesion molecule for intracellular adhesion molecule, is also increased during active disease [77]. Soluble VCAM levels have been found to be decreased by treatment with natalizumab and recover in patients who develop neutralizing antibodies to natalizumab [78]. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes that dissolve the extracellular matrix and assist in leukocyte migration through tissue during inammation. MMP-9 in particular has been found to be elevated in serum during MS relapse, and levels

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of its inhibitor TIMP-1 also decline during disease activity [79,80]. IFN treatment decreases MMP-9 levels and correlates with improvements in disease activity [81]. MMP-8 levels also appear to correlate with disease activity [19]. 9. Chemokines Chemokines and their ligands also help target different immune cell subsets to areas of inammation (Table 6). Serum levels of CXCL8, CXCR3, CCL5 and IP-10 increase during relapse with MRI activity, while levels of CCL2 (MCP1) decline [8284]. CCL2 and CXCL10 levels are strongly induced by IFN- treatment and lost in the presence of IFN- neutralizing antibodies, but their correlation with disease activity is unknown [85]. Serum levels of the chemokine IL-8 in the rst trimester of pregnancy have been shown to predict patients at risk of post-partum relapse [86]. Serum levels of the CD8+ T cell chemokine receptor CXCR3 correlate with MRI measures of CNS damage and clinical relapse [87,88]. Serum levels of the B cell chemokine CXCL13 are elevated during active MS, but appear to be unaffected by IFN- or glatiramer acetate therapy [89]. Monocyte expression of CCR5 also has shown a minor correlation with disease activity in MS [90]. The NK cell chemokine CX3CR1 is decreased during active relapse [91,92]. Genetic polymorphisms in CCL5 and CCR5 have been found to correlate with clinical and MRI measures of disease severity [92]. 10. Other markers Several other biomarkers have been explored in MS (Tables 7 and 8). Myxovirus resistance protein A (MxA) is produced by a variety of cell types in response to type I interferons (IFN- and IFN-). Lower levels of MxA mRNA in blood prior to any treatment correlate with ongoing clinical and MRI disease activity and predict subsequent relapse rate independently of the presence of MRI contrast-enhancing lesions [93]. Induction of MxA protein expression by IFN- treatment has been variably reported to correlate with clinical response to treatment [94]. MxA probably also serves as an indirect measure of IFN- bioavailability, as MxA levels decline in patients who develop high titer IFN- neutralizing antibodies (NAbs), and correlate with later relapses [95]. Recent attention has focused on the role that Vitamin D plays in multiple sclerosis. Higher levels of 25-hydroxyVitamin D are predictive of relapse rates in the next 6 months [96]. Brain derived neurotrophic factor (BDNF) is produced by a variety of cell types including immune cells during tissue repair. BDNF production by monocytes is stimulated by IFN- and glatiramer and may play a role in promoting tissue repair in the CNS after damage, as higher BDNF production correlates with better recovery after relapse [9799]. Additional growth factors (glial derived neurotrophic factor, neural growth factor, and neurotrophins 3 and 4) have also been found to correlate with recovery from relapses [99]. Serum levels of uric acid, which acts as a scavenger of nitric oxide products, decline during active disease and improve with remission, though others failed to conrm these ndings [100,101]. Pentosidine is an advanced glycation end product that accumulates in plasma after tissue damage by oxidation or inammation in various disease states. Plasma pentosidine levels have recently been described to correlate with disability as measured by EDSS and MSFC, though it did not correlate with relapse rates [102]. Neopterin is a catabolic product of guanine triphosphate produced by stimulated macrophages, serving as a marker of inammatory activity. Stable urinary neopterin/creatinine ratios correlated with disease stability in a trial of IFN- in patients with primary progressive MS [103]. Numerous genetic markers of MS risk and disease course have been explored with potential utility as biomarkers of disease course and possibly application to therapeutic decisions [104].

11. CSF biomarkers The most commonly used biomarker in MS remains CSF oligoclonal bands (OCB). The presence of OCB in the CSF greatly increases the likelihood that an individual presenting with an initial monophasic, clinically isolated demyelinating syndrome will eventually experience a second episode, meeting clinical criteria for the diagnosis of RRMS, independent of MRI ndings [105]. Patients without CSF OCB have been reported to have a less aggressive course of disease activity [105]. It should be kept in mind that some methods of isolating CSF OCB are superior to others, with some methods reaching sensitivity of 90%, while other techniques less frequently to detect CSF OCB [105]. Therefore, results of CSF OCB testing should be considered in the context of the method used. The absence of CSF OCB certainly does not exclude the possibility of MS, but should raise concern for an alternate diagnosis. Conversely, CSF OCB are not specic and can be found less frequently in numerous other conditions that can clinically mimic MS including various inammatory, infectious and neoplastic conditions, though usually OCB will only transiently be present in these conditions, whereas they are continuously present in MS patients regardless of disease activity [105]. The presence of IgM OCB has been reported to correlate with subsequent disability progression [106]. Recently OCB have been detected in the tears of patients with CIS with 100% specicity but less sensitivity than CSF [107]. The presence of mature B cells or plasma blasts (CD19+CD138+) in the CSF correlate with MRI activity [55]. Elevated kappa free light chains in the CSF appear to predict both MRI and clinical disease activity [108,109]. Antibody responses against myelin and other CNS lipid antigens (sulfatide, ganglioside, etc.) have also been found in the CSF of MS patients and their severity predicted MRI response in a phase 2 trial of DNA vaccination in concert with a reduction in CSF antibody responses to myelin [110,111]. Humoral activation in the CSF may play an important role in disease progression, as chronic B cell activation in ectopic lymph tissue of the meninges appears to contribute to gray matter damage in MS, especially in patients with progressive forms [112]. CSF levels of IL-12p40 and IL-6 increase during active disease [16,113]. CSF IL-10 levels increase after treatment with IFN- which correlates with clinical response [31]. Mononuclear cells producing IL-17 increase in CSF during relapse [23]. CSF levels of TNF- are increased in active MS and correlate with risk of future disability [70,71]. Osteopontin levels in the CSF also appear to correlate with disease activity [114]. In NMO, there is some evidence that CSF IL-6 levels may correlate with disability, relapses and disease severity [115]. The percentage of nave lymphocytes expressing ICAM-3 in CSF is increased during active relapse [116]. CSF levels of sICAM and sVCAM were elevated during disease activity and showed an inverse relationship with distance of the active lesions from the ventricular surface [117]. Levels of the T and B cell chemokine CXCL13 (ligand for CXCR5) in CSF correlate with ongoing disease activity and decline after treatment with methylprednisolone or natalizumab [118,119]. Levels of CXCL12 and IP-10 in the CSF are also elevated during active disease, while levels of the monocyte chemoattractant protein CCL2 decline [82,113,118]. Levels of CCL5 in CSF are increased during active disease but decline during periods of remission [120]. Several proteins that serve as markers of ongoing tissue damage and repair have also been found in the CSF in patients with MS. Nitrite, nitrate and nitric oxide degradation products are increased in CSF during active relapse and correlate with lesion size and relapse duration [121,122]. In a study of patients with CIS, those with higher levels of the protein chitinase 3-like 1 had more MRI lesions and higher likelihood of converting to clinically denite MS and accumulating xed disability during follow-up [123]. Immune responses play a role in repair after injury by release of neurotrophic factors [9]. CSF levels of neuronal cell adhesion molecule (NCAM) and

J.J. Graber, S. Dhib-Jalbut / Journal of the Neurological Sciences 305 (2011) 110

ciliary neurotrophic factor (CNTF) are elevated during relapse and correlate with clinical recovery [124]. Glial activation during active relapse is detectable in the CSF by elevated levels of S-100 and CSF levels of glial brillary acidic protein rise over time with progressive disability [125,126]. Various non-specic markers of neuronal damage in CSF can be found during active disease, including light and heavy neurolament chains, 14-3-3 protein and tau protein [127,128]. CSF levels of neurolament light chain, a structural intracellular protein of neurons released when neurons are damaged, increase during relapse, and correlate with long term functional outcomes [129]. Higher levels of neurolament heavy chains in the CSF increase the likelihood of conversion from CIS to RRMS and also predict greater future disability [128130]. N-acetyl aspartate levels in the CSF are higher in RRMS than SPMS and correlate with more active disease [131]. CSF levels of MBP correlate with active myelin breakdown, but are not specic to MS, and decline in concert with clinical recovery and disappearance of gadolinium enhancement after treatment with methylprednisolone [132,133]. CSF levels of the transmembrane neuronal protein Bri2-23 are specically decreased in SPMS patients and correlate with clinical measures of cerebellar and cognitive function [134]. CSF levels of Fetuin-A, an immunoregulatory protease inhibitor, appear to be specically elevated in active secondary progressive (but not relapsing remitting or primary progressive) MS [134]. Though initially reported to be remarkably specic for MS, the presence of soluble Nogo-A, an inhibitor of neuronal axon outgrowth, was later refuted to be a useful CSF biomarker of MS [135,136]. 12. Conclusions Biomarkers of disease activity may eventually assist in predicting disease course, responsiveness to different therapeutic options, and guide changes in therapy for patients with multiple sclerosis. It is necessary to consider that multiple factors play complex roles in MS pathogenesis and treatment across the lifespan of the patient (see Fig. 2). Recently described biomarkers are helping to elucidate the complex and heterogenous pathophysiology of MS and show promise for future clinical applications (Table 9). Advances in MR technology may help to better predict outcomes and guide therapy, for example, elevated brain myoinositol has been found to correlate with later cognitive dysfunction [3,137]. Currently identied and validated biomarkers are useful in initial diagnosis of MS versus NMO (aquaporin-4 antibody), predicting conversion to RRMS after CIS (CSF OCB) and interpreting treatment failure in patients on IFN-

Table 9 Recently identied potential biomarkers. OCB in tears K2P5.1 CCL2 CXCL10 IL-8 Complement C4 fragment Complement regulatory factor H miRNAs altering CD47 expression MMP-8 Corresponds to intrathecal OCB Potassium channel affecting T cells Chemokine Chemokine Chemokine Marker of complement activation Marker of complement activity [107] [45] [85,119] [85,119] [86] [66] [63] [75] [19] [10,67,68] [96] [103] [63] [137] [134] [134] [102] [143]

Alters vulnerability to macrophage phagocytosis Affects inammatory cell migration through tissue CD56bright NK cells Regulate activated T cells 25-Hydroxy-vitamin Has immunomodulatory effects D Urinary neopterin Marker of macrophage activity ILT3 Downregulates immune activity Myoinositol MR marker of tissue integrity Bri2-23 Neuronal protein Fetuin-A Immunoregulatory protease inhibitor Pentosidine Marker of tissue damage and inammation Leptin receptor Metabolic and immune pathways IL = interleukin. ILT = immunoglobulin-like transcript. OCB = oligoclonal bands. MMP = matrix metalloprotease.

therapy (IFN- neutralizing antibodies), but are clearly not infallible (Table 10). It is possible that multiplex arrays and statistical algorithms that examine multiple biomarkers simultaneously may provide better sensitivity and specicity [10,62,85,138142]. Reliable biomarkers of disease activity and therapeutic response would assist in guiding treatment decisions and improving long-term outcomes in MS.

Table 10 Biomarkers associated with various disease stages and clinical phenotypes. Biomarker CSF OCB CD19+ B cells in CSF CD39+ Treg CXCL3 BDNF (CSF) Fetuin A CIS Predicts RRMS Common RRMS Present in 90% Common SPMS PPMS NMO

Absent

Less Less frequent frequent Absent

Elevated

Predictive profiles of disease phenotypes and treatment response

Decreased Normal Elevated Normal Highest Elevated Active SPMS

Normal Reduced

Immunology & Genetics

Pathology & Imaging

Neurolament Predicts chains (CSF) RRMS NAA (CSF) Vitamin D binding protein Neopterin (urine) Jagged-1 (CSF) Aquaporin-4 antibody

Increased High

Lower Low High if stable High Present in 6080%

Normal Predicts NMO Absent in LETM, severe or bilateral ON

Clinical Phenotypes

Fig. 2. Predictive proles of disease phenotypes and treatment response.

BDNF = brain derived neurotrophic factor. CIS = clinically isolated syndrome. CSF = cerebrospinal uid. LETM = longitudinally extensive transverse myelitis. NMO = neuromyelitis optica. OCB = oligoclonal bands. ON = optic neuritis. PPMS = primary progressive multiple sclerosis. RRMS = relapsing remitting multiple sclerosis. SPMS = secondary progressive multiple sclerosis. Treg = regulatory T cells.

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