# Biomarkers of multiple sclerosis

Several biomarkers for diagnosis of multiple sclerosis, disease evolution and response to medication (current or expected) are under research. While most of them are still under research, there are some of them already well stablished:
* oligoclonal bands: They present proteins that are in the CNS or in blood. Those that are in CNS but not in blood suggest a diagnosis of MS.
* MRZ-Reaction: A polyspecific antiviral immune response against the viruses of measles, rubella and zoster found in 1992. In some reports the MRZR showed a lower sensitivity than OCB (70% vs. 100%), but a higher specificity (69% vs. 92%) for MS.
* free light chains (FLC). Several authors have reported that they are comparable or even better than oligoclonal bands.
They can be of several types like body fluid biomarkers, imaging biomarkers or genetic biomarkers. They are expected to play an important role in the near future of MS.

## Classification

Biomarkers can be classified according to several criteria. It is common to classify them according to their source (imaging biomarkers, body fluid biomarkers and genetic biomarkers) or their utility (diagnosis, evolution and response to medication)
Among the imaging biomarkers in MS the most known is MRI by two methods, gadolinium contrast and T2-hypertense lesions, but also important are PET and OCT.
Among the body fluid biomarkers the most known are oligoclonal bands in CSF but several others are under research.
Genetic biomarkers are under study but there is nothing conclusive still.
Addressing the classification by its utility we have diagnosis biomarkers, evolution biomarkers and response to medication biomarkers.

## Biomarkers for diagnosis

Apart from its possible involvement in disease pathogenesis, vitamin D has been proposed as a biomarker of the disease evolution.
Diagnosis of MS has always been made by clinical examination, supported by MRI or CSF tests. According with both the pure autoimmune hypothesis and the immune-mediated hypothesis, researchers expect to find biomarkers able to yield a better diagnosis, and able to predict the response to the different available treatments.
As of 2016 no specific biomarker for MS has been found, but several studies are trying to find one. Some researchers are focusing also in specific diagnosis for each of the clinical courses
Some people focus on blood tests, given the easy availability for diagnosis. Among the studies for blood tests, the highest sensitivity and specificity reported to date is testing circulating erythrocytes (s=98.3%, e=89.5%). Also a good result was obtained using methylation patterns of circulating cell debris are specific for a number of conditions, including RRMS There are ongoing efforts to be able to diagnose MS by analysing myelin debris into the blood stream.
As of 2014, the only fully specific biomarkers found were four proteins in the CSF: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin-T (a phosphodiesterase) This list was expanded in 2016, with three CSF proteins (Immunoglobulins) reported specific for MS. They are the following immunoglobulins: Ig γ-1 (chain C region), Ig heavy chain V-III (region BRO) and Ig-κ-chain (C region)

## For existing damage and disease evolution

During a clinical trial for one of the main MS drugs, a catheter was inserted into the brain's ventricles of the patients. Existing damage was evaluated and correlated with body fluids. Thanks to the courage of these volunteers, now we know that in PPMS, neurofilament light chain (NF-L) level, in CSF and serum, is a sensitive and specific marker for white matter axonal injury
About biomarkers for MRI images, Radial Diffusivity has been suggested as a biomarker associated with the level of myelination in MS lesions. However, it is affected also by tissue destruction, which may lead to exaggeration of diffusivity measures. Diffusivity can be more accurate. Distinct patterns of diffusivity in MS lesions suggest that axonal loss dominates in the T1 hypointense core and that the effects of de/remyelination may be better detected in the "T2-rim", where there is relative preservation of structural integrity.
Glial fibrillary acidic protein (GFAP) has been indicated as a possible biomarker for the progression of MS. The blood level of GFAP increases when astrocytes are damaged or activated, and elevated levels of the protein's cellular component correlate with severity of MS symptoms.

## Treatments and response to therapy

Currently the only clear biomarker that predicts a response to therapy is the presence of anti-MOG autoantibodies in blood. Anti-MOG seropositive patients do not respond to approved MS medications. In fact, it seems that MS patients with anti-MOG positivity could be considered a different disease in the near future.
Comparative Effectiveness Research (CER) is an emerging field in Multiple Sclerosis treatment. The response of the disease to the different available medications at this moment cannot be predicted, and would be desirable.
But the ideal target is to find subtypes of the disease that respond better to a specific treatment. A good example could be the discovery that the presence of a gene called SLC9A9 appears in people who fail to respond to interferon β therapy or that the disregulation of some transcription factors define molecular subtypes of the disease Other good example could be the Hellberg-Eklund score for predicting the response to Natalizumab.
Though biomarkers are normally assumed to be chemical compounds in body fluids, image can also be considered a biomarker. For an example about research in this area, it has been found that fingolimod is specially suitable for patients with frequently relapsing spinal cord lesions with open-ring enhancement. Anyway, patients with spinal cord lesions could have different T-helper cells patterns that those with brain lesions.
Biomarkers are also important for the expected response to therapy. As an example of the current research, in 2000 was noticed that patients with pattern II lesions were dramatically responsive to plasmapheresis, and in February 2016, it was granted the first patent to test the lesion pattern of a patient without biopsy.
Other examples could be the proposal for protein SLC9A9 (gen Solute carrier family 9) as biomarker for the response to interferon beta, as it happens for serum cytokine profiles The same was proposed to MxA protein mRNA. The presence of anti-MOG, even with CDMS diagnosis, can be considered as a biomarker against MS disease modifying therapies like fingolimod.
Diagnosis of MS has always been made by clinical examination, supported by MRI or CSF tests. According with both the pure autoimmune hypothesis and the immune-mediated hypothesis, researchers expect to find biomarkers able to yield a better diagnosis, and able to predict the response to the different available treatments. As of 2014 no biomarker with perfect correlation has been found, but some of them have shown a special behavior like IgG- and IgM- oligoclonal bands in the cerebrospinal fluid and autoantibodies against neurotropic viruses (MRZ reaction) and the potassium channel Kir4.1.
A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention. Type 0 biomarkers are those related to the course a pathogenic process and type 1 are those that show the effects of the therapeutical intervention.
As of 2014, the only fully specific biomarkers found to date are four proteins in the CSF: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin-T (a phosphodiesterase). Nevertheless, abnormal concentrations of non-specific proteins can also help in the diagnosis, like chitinases. This list has been expanded in 2016, with three CSF proteins (Immunoglobulins) reported specific for MS. They are the following immunoglobulins: Ig γ-1 (chain C region), Ig heavy chain V-III (region BRO) and Ig-κ-chain (C region).
Biomarkers are also important for the expected response to therapy. Currently it has been proposed the protein SLC9A9 (gen Solute carrier family 9) as biomarker for the response to interferon beta.

### Molecular biomarkers in blood

Blood serum of MS patients shows abnormalities. Endothelin-1 shows maybe the most striking discordance between patients and controls, being a 224% higher in patients than controls.
Creatine and Uric acid levels are lower than normal, at least in women. Ex vivo CD4(+) T cells isolated from the circulation show a wrong TIM-3 (Immunoregulation) behavior, and relapses are associated with CD8(+) T Cells. There is a set of differentially expressed genes between MS and healthy subjects in peripheral blood T cells from clinically active MS patients. There are also differences between acute relapses and complete remissions. Platelets are known to have abnormal high levels.
MS patients are also known to be CD46 defective, and this leads to Interleukin-10 (IL-10) deficiency, being this involved in the inflammatory reactions. Levels of IL-2, IL-10, and GM-CSF are lower in MS females than normal. IL6 is higher instead. These findings do not apply to men. This IL-10 could be related to the mechanism of action of methylprednisolone, together with CCL2. Interleukin IL-12 is also known to be associated with relapses, but this is unlikely to be related to the response to steroids.
Kallikreins are found in serum and are associated with secondary progressive stage. Related to this, it has been found that B1-receptors, part of the kallikrein-kinin-system, are involved in the BBB breakdown.
There is evidence of Apoptosis-related molecules in blood and they are related to disease activity. B cells in CSF appear, and they correlate with early brain inflammation. There is also an overexpression of IgG-free kappa light chain protein in both CIS and RR-MS patients, compared with control subjects, together with an increased expression of an isoforms of apolipoprotein E in RR-MS. Expression of some specific proteins in circulating CD4+ T cells is a risk factor for conversion from CIS to clinically defined multiple sclerosis.
Recently, unique autoantibody patterns that distinguish RRMS, secondary progressive (SPMS), and primary progressive (PPMS) have been found, based on up- and down-regulation of CNS antigens, tested by microarrays. In particular, RRMS is characterized by autoantibodies to heat shock proteins that were not observed in PPMS or SPMS. These antibodies patterns can be used to monitor disease progression.
Finally, a promising biomarker under study is an antibody against the potassium channel protein KIR4.1. This biomarker has been reported to be present in around a half of MS patients, but in nearly none of the controls.

### Micro-RNA in blood

Micro-RNA are non-coding RNA of around 22 nucleotides in length. They are present in blood and in CSF. Several studies have found specific micro-RNA signatures for MS. They have been proposed as biomarkers for the presence of the disease and its evolution and some of them like miR-150 are under study, specially for those with lipid-specific oligoclonal IgM bands
Circulating MicroRNAs have been proposed as biomarkers. There is current evidence that at least 60 circulating miRNAs would be dysregulated in MS patient's blood and profiling results are continuously emerging. Circulating miRNAs are highly stable in blood, easy to collect, and the quantification method, if standardized, can be accurate and cheap. They are putative biomarkers to diagnose MS but could also serve differentiating MS subtypes, anticipating relapses and proposing a customized treatment. MiRNA has even been proposed as a primary cause of MS and its white matter damaged areas

### Genetic biomarkers for MS type

    By RNA profile
    Also in blood serum can be found the RNA type of the MS patient. Two types have been proposed classifying the patients as MSA or MSB, allegedly predicting future inflammatory events.
    By transcription factor
    The autoimmune disease-associated transcription factors EOMES and TBX21 are dysregulated in multiple sclerosis and define a molecular subtype of disease. The importance of this discovery is that the expression of these genes appears in blood and can be measured by a simple blood analysis.
    NR1H3 Mutation.
    Some PPMS patients have been found to have a special genetic variant named rapidly progressive multiple sclerosis In these cases MS is due to a mutation inside the gene NR1H3, an arginine to glutamine mutation in the position p.Arg415Gln, in an area that codifies the protein LXRA.

### In blood vessel tissue

Endothelial dysfunction has been reported in MS and could be used as biomarker via biopsia. Blood circulation is slower in MS patients and can be measured using contrast or by MRI
Interleukin-12p40 has been reported to separate RRMS and CIS from other neurological diseases

### In cerebrospinal fluid

The most specific laboratory marker of MS reported to date, as of 2016, is the intrathecal MRZ (Measles, Rubella and Varicella) reaction showing 78% sensitivity and 97% specificity.
It has been known for quite some time that glutamate is present at higher levels in CSF during relapses, maybe because of the IL-17 disregulation, and to MS patients before relapses compared to healthy subjects. This observation has been linked to the activity of the infiltrating leukocytes and activated microglia, and to the damage to the axons and to the oligodendrocytes damage, supposed to be the main cleaning agents for glutamate
Also a specific MS protein has been found in CSF, chromogranin A, possibly related to axonal degeneration. It appears together with clusterin and complement C3, markers of complement-mediated inflammatory reactions. Also Fibroblast growth factor-2 appear higher at CSF.
Varicella-zoster virus particles have been found in CSF of patients during relapses, but this particles are virtually absent during remissions. Plasma Cells in the cerebrospinal fluid of MS patients could also be used for diagnosis, because they have been found to produce myelin-specific antibodies. As of 2011, a recently discovered myelin protein TPPP/p25, has been found in CSF of MS patients
A study found that quantification of several immune cell subsets, both in blood and CSF, showed differences between intrathecal (from the spine) and systemic immunity, and between CSF cell subtypes in the inflammatory and noninflammatory groups (basically RRMS/SPMS compared to PPMS). This showed that some patients diagnosed with PPMS shared an inflammatory profile with RRMS and SPMS, while others didn't.
Other study found using a proteomic analysis of the CSF that the peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients
As of 2014 it is considered that the CSF signature of MS is a combination of cytokines CSF lactate has been found to correlate to disease progression
Three proteins in CSF have been found to be specific for MS. They are the following immunoglobulins: Ig γ-1 (chain C region), Ig heavy chain V-III (region BRO) and Ig-κ-chain (C region)
Other interesting byproduct of the MS attack are the neurofilaments, remainings of the neural damage and the immunoglobulin heavy chains.

### Oligoclonal bands

CSF also shows oligoclonal bands (OCB) in the majority (around 95%) of the patients. Several studies have reported differences between patients with and without OCB with regard to clinical parameters such as age, gender, disease duration, clinical severity and several MRI characteristics, together with a varying lesion load. CSF oligoclonal bands can be reflected in serum or not. This points to a heterogeneous origin of them
Though early theories assumed that the OCBs were somehow pathogenic autoantigens, recent research has shown that the immunoglobulins present in them are antibodies against debris, and therefore, OCBs seem to be just a secondary effect of MS.
Given that OCBs are not pathogenic, their remaining importance is to demonstrate the production of intrathecal immunoglobins (IgGs) against debris, but this can be shown by other methods. Specially interesting are the free light chains (FLC), specially the kappa-FLCs (kFLCs). Free kappa chains in CSF have been proposed as a marker for MS evolution

### Biomarkers in brain cells and biopsies

Abnormal sodium distribution has been reported in living MS brains. In the early-stage RRMS patients, sodium MRI revealed abnormally high concentrations of sodium in brainstem, cerebellum and temporal pole. In the advanced-stage RRMS patients, abnormally high sodium accumulation was widespread throughout the whole brain, including normal appearing brain tissue. It is currently unknown whether post-mortem brains are consistent with this observation.
The pre-active lesions are clusters of microglia driven by the HspB5 protein, thought to be produced by stressed oligodendrocytes. The presence of HspB5 in biopsies can be a marker for lesion development.
Retinal cells are considered part of the CNS and present a characteristic thickness loss that can separate MS from NMO

### Biomarkers for the clinical course

Currently it is possible to distinguish between the three main clinical courses (RRMS, SPMS and PPMS) using a combination of four blood protein tests with an accuracy around 80%
Currently the best predictor for clinical multiple sclerosis is the number of T2 lesions visualized by MRI during the CIS, but it has been proposed to complement it with MRI measures of BBB permeability It is normal to evaluate diagnostic criteria against the "time to conversion to definite".

## Imaging biomarkers: MRI, PET and OCT

Magnetic resonance (MRI) and positron emission tomography (PET) are two techniques currently used in MS research. While the first one is routinely used in clinical practice, the second one is also helping to understand the nature of the disease.
In MRI, some post-processing techniques have improved the image. SWI-adjusted magnetic resonance has given results close to 100% specificity and sensitivity respect McDonald's CDMS status and magnetization transfer MRI has shown that NAWM evolves during the disease reducing its magnetization transfer coefficient.
PET is able to show the activation status of microglia, which are macrophage-like cells of the CNS and whose activation is thought to be related to the development of the lesions. Microglial activation is shown using tracers for the 18 kDa translocator protein (TSPO) like the radioligand $^{11}$ [C]PK11195

## Biomarkers for MS pathological subtype

Differences have been found between the proteins expressed by patients and healthy subjects, and between attacks and remissions. Using DNA microarray technology groups of molecular biomarkers can be established. For example, it is known that Anti-lipid oligoclonal IgM bands (OCMB) distinguish MS patients with early aggressive course and that these patients show a favourable response to immunomodulatory treatment.
It seems that Fas and MIF are candidate biomarkers of progressive neurodegeneration. Upregulated levels of sFas (soluble form of Fas molecule) were found in MS patients with hypotensive lesions with progressive neurodegeneration, and also levels of MIF appeared to be higher in progressive than in non-progressing patients. Serum TNF-α and CCL2 seem to reflect the presence of inflammatory responses in primary progressive MS.
As previously reported, there is an antibody against the potassium channel protein KIR4.1 which is present in around a half of MS patients, but in nearly none of the controls, pointing towards a heterogeneous etiology in MS. The same happens with B-Cells

### DRB3*02:02 patients

Specially interesting is the case of DRB3*02:02 patients (HLA-DRB3*–positive patients), which seem to have a clear autoimmune reaction against a protein called GDP-L-fucose synthase.

## Biomarkers for response to therapy

Response to therapy is heterogeneous in MS. Serum cytokine profiles have been proposed as biomarkers for response to Betaseron and the same was proposed to MxA mRNA.