juvenile idiopathic arthritis ( jia ) is the most common inflammatory rheumatic disease in childhood , affecting one in 1000 children .
jia is characterized by severe joint inflammation in one or more joints , which persists for at least six weeks , with disease onset before the age of 16 .
this heterogeneous group of diseases can be divided into several subtypes on the basis of clinical symptoms , medical history , and abnormalities in laboratory measures .
a biomarker is a small component which is easily measurable in accessible patient material , e.g. blood , urine or saliva , and is ideally obtained using a relatively non - invasive approach .
ideally , the component used as a biomarker should be stable over time within the sample , and would be able to be measured by an accurate , reproducible assay , at a relatively affordable cost to health service providers .
in addition , the ideal biomarker for paediatric use would not be affected by age - related development of children , avoiding the need for age - specific normal - range data sets .
biomarkers are already used in many areas of clinical practice , but most biomarker studies focus on adults rather than children .
data from these studies are sometimes extrapolated to children without considering differences in disease pathogenesis , age - dependent changes in reference ranges for biological laboratory measures , growth and development of children over time , effect of ontogeny on disease evolution and response to treatment , and changes in phenotypic gene expression [ 3 , 4 ] . despite the huge potential of paediatric biomarkers , for jia
there are currently no validated paediatric biomarkers available to help in setting up a tailored or
a more tailored approach would be beneficial for patients because it could facilitate disease remission at an earlier disease stage , which would reduce burden of disease , limit side effects , and improve quality of life . in this review
we will discuss recent developments in the potential use of biomarkers for jia , for predicting the type , severity , and progression of disease after onset and the development of complications linked to jia , and their effect on our ability to predict response to treatment and determine when stable disease remission has been reached .
jia is a heterogeneous group of disorders , and its classification relies on both clinical findings and a small number of biomarkers used to divide cases into relatively homogeneous subtypes .
for example , the two polyarticular forms of jia , involving five or more joints in the first six months of disease , are distinguished by the absence or presence of serum autoantibody , known as rheumatoid factor ( rf ) , on two occasions three months or more apart .
these two clinical subgroups of jia are distinct in their genetics , age of onset , and prognosis [ 6 , 7 ] .
similarly , the presence of positive serum anti - nuclear antibody ( ana ) has been revealed in several studies to be associated with an increased risk of chronic anterior uveitis in jia .
this is a serious comorbidity of jia , involving painless but potentially very damaging inflammation of the anterior chamber of the eye , which requires assiduous screening to prevent permanent visual loss [ 8 , 9 ] .
a recent large cohort study has confirmed earlier studies demonstrating that ana positivity is a risk factor for developing jia - associated uveitis ; current uk clinical guidelines for how long screening should be continued include the use of ana positivity to direct clinical practice .
thus , both of these antibody biomarkers ( rf and ana ) , which are stable proteins and are easily measured in a small volume of serum , form part of routine clinical care and treatment pathway decisions for jia . at the mild end of the jia clinical spectrum ,
oligoarticular jia , which presents with involvement of four or fewer joints in the first six months of disease , can lead to widely divergent outcomes , ranging from complete remission off medication , to a more severe , extended form of jia which spreads to involve many joints .
extended oligoarticular jia can be highly erosive and destructive and may be difficult to control with conventional disease - modifying antirheumatic drugs ( dmards ) , frequently requiring long - term treatment with biological therapy .
several studies have revealed that immunological differences between these two outcomes ( persistent versus extended oligoarticular jia ) can be observed in the inflamed joint .
for example , t cell types ( e.g. regulatory t cells , or highly proinflammatory th17 cells ) and their frequencies differ significantly between these two clinical types [ 13 , 14 ] . in addition , differences in cell frequencies , inflammatory protein levels , and gene expression can be detected in children who will develop more severe disease , before extension occurs : for example , in the so - called  extended - to - be  group of cases , the cd4:cd8 ratio in synovial fluid is lower and the levels of the chemokine ccl5 are higher in extended - to - be oligoarticular jia compared with persistent oligoarticular jia [ 15 ] . in this study ,
analysis of differentially expressed genes at mrna level also provided novel insights into the pathological mechanisms involved in severity , indicating the importance of the complement pathway and of activated monocytes in extension to more severe disease .
studies analysing the proteome within synovial fluid have also revealed differences between these subtypes : gibson et al . demonstrated that the proteome and post - translational modifications of proteins differed between those whose disease remained mild and those who went on to develop severe disease .
validation of these findings will be required before development of a predictive biomarker test , but this field may well yield valuable biomarkers by which to identify children with a poor prognosis .
assessment of disease activity in jia typically includes measurement of inflammatory markers in peripheral blood , including either the erythrocyte sedimentation rate ( esr ) or c - reactive protein ( crp ) [ 17 , 18 ] .
both of these inflammatory indices have low sensitivity and specificity , because they can be raised for many reasons other than jia activity and in some children do not closely mirror disease activity .
recently , the pro - inflammatory s100 proteins , s100a8/9 ( also known as calprotectin or myeloid - related protein ( mrp ) 8/14 ) and s100a12 , have been described to be sensitive measures for disease activity in jia , and both correlate well with physicians assessment of disease or with actively inflamed joints [ 5 , 19 ] .
s100 proteins can be measured in serum by enzyme - linked immune - sorbent assay ( elisa ) . however , the standardisation of the measurements and the detailed procedures for assay and dilution of serum samples from a wide range of patients , with varying disease activity , are non - trivial , complicating implementation of such a test in routine clinical care .
the s100 proteins are released into serum at highly elevated levels in the most severe form of jia , systemic onset jia ( sjia ) , where they correlate well with disease activity  as assessed by physicians global assessment of disease activity ( r  =  0.62 ) , childhood health assessment questionnaire ( r  =  0.56 ) , and active joint count ( r  =  0.46)and with crp ( r  =  0.71 ) and esr ( r  =  0.72 ) ( for all p  <  0.001 ) .
a recent study which included measurement of serum s100 proteins has proposed a biomarker panel for predicting flare in sjia , compared with quiescent disease .
the same group also tested the feasibility of measuring such biomarkers in urine for sjia , which could have many advantages for the paediatric population , especially if testing  kits  could be designed for use in the clinic office or even at home [ 22 ] .
one of the most severe complications of sjia , known as macrophage activation syndrome ( mas ) or secondary hemophagocytic lymphohistiocytosis ( hlh ) , remains a cause of mortality in jia and may be difficult to distinguish from infection [ 23 , 24 ] . a gene expression profiling study , of sjia patients with or without mas compared with control patients , identified clusters of genes that correlated with sjia activity and with mas .
if validated , these gene clusters could lead to an mrna - expression biomarker panel for predicting this potentially life - threatening complication , or to distinguish it from other complications .
interestingly , some of the differentially expressed mrna species identified , which differed between children with mas and those without , were transcripts of genes known to be involved in other causes of hlh , for example rab27a and sh2d1a .
thus , several autoantibodies are already in widespread use as biomarkers in routine care of jia , and some newer biomarkers ( protein , cellular , or mrna ) are under development , for use in defining disease subtype , probable disease course , or comorbidity .
recommendations for treatment of jia are made by the american college of rheumatology ( acr ) , and are regularly reviewed .
depending on the number of joints affected  and after failure of monotherapy with non - steroidal anti - inflammatory drugs ( nsaids ) and/or glucocorticoids joint injection in cases of less severe disease  the first - line treatment approach for active disease is administration of methotrexate ( mtx ) . in cases of no or poor response to mtx ,
biological agents , for example drugs which block tumour necrosis factor alpha ( tnf ) , are added to the treatment strategy .
although many patients respond well to mtx and reach stable disease remission , approximately 3050  % [ 2729 ] of patients treated with mtx either do not respond or respond poorly .
many studies analysing response to mtx used the core set variables defined by giannini et al .
, in which levels of response in terms of acr30 , 50 , or 70 are calculated .
those patients who fail to respond to mtx are first exposed to mtx , and experience side effects associated with non - response to mtx , before more effective biological agents can be offered .
a biomarker ( or set of biomarkers ) predicting which patients will respond to treatment would be beneficial for the patient and prevent side effects caused by ineffective drugs .
the pro - inflammatory s100 proteins have been shown to correlate well with disease activity [ 5 , 19 , 20 , 30 ] , and recently the level of s100 proteins in serum has been shown to also be correlated with response to treatment .
six months after starting treatment , mrp8/14 levels were lower in sjia patients responding to mtx treatment ( defined as reaching at least acr70 ) , whereas mrp8/14 levels in sjia patients not responding to mtx treatment ( acr30 ) were increased or slightly decreased .
furthermore , sjia patients treated with anti - il1 or anti - tnf had reduced mrp8/14 levels and disease activity .
patients who did respond to mtx had more active joints , higher crp levels , and higher serum cytokine levels before the start of mtx , which suggests that higher disease activity is positively correlated with good response to mtx .
in addition , jia patients with high levels of mrp8/14 before starting mtx treatment had a higher chance of good response to mtx and had better disease outcomes ( at least acr50 ) after six months of treatment than non - responders ( acr30 or below ) .
differences between patients and response to treatment could be genetic , and therefore genetic factors could be potential biomarkers .
one study identified two single nucleotide polymorphisms ( snps ) in the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase ( atic ) gene and one snp in the inosine triphosphate pyrophosphatase ( itpa ) gene which were found to be associated with a higher risk of poor response to mtx treatment . using a validation cohort
, one of the snps in the atic gene had a trend towards association with response to mtx .
genotyping of snps in genes involved in the polyglutamylation process of mtx and in cellular uptake and efflux of mtx have also shown association with mtx response .
one snp in the adenosine - triphosphate - binding cassette transporter b1 ( abcb1 ) and one snp in abcc3 were found to be associated with good response to mtx , whereas an snp in the solute carrier 19a1 was associated with poor response to mtx . in another approach ,
snp genotyping of genes found to be differentially expressed in a gene expression profiling study , of a uk cohort of jia patients before and after treatment with mtx , identified three snps in the solute carrier family 16 member 7 ( slc16a7 ) gene associated with response to mtx .
validation of these snps in a validation cohort revealed significant association of one of the snps with non - response to mtx .
a prediction model was recently developed combining clinical and genetic variables to predict non - response of jia patients to mtx . in this study ,
response was defined as reaching acr70 in at least two out of three visits during the first year of treatment .
such a model is important , because it could help in preventing unnecessary treatment of patients who will benefit from monotherapy with mtx and do not need additional treatment with biologicals , which are expensive and might have side effects . the clinical variable included in the model is the esr . genetic variables are snps in the genes coding for methionine synthase reductase , multidrug resistance 1 ( mdr-1/abcb1 ) , multidrug resistance protein 1 ( mrp-1/abcc1 ) , and proton - coupled folate transporter ( pcft ) .
the model had a moderate predictive power of 65  % for the validation cohort , which might be because of the small number of patients included in the validation cohort [ 34 ] .
thus , evidence suggests that the pro - inflammatory s100 proteins have the potential to serve as biomarkers for predicting response to treatment .
furthermore , several snps have been found to be associated with response to treatment , indicating that genetic factors can also provide such biomarkers .
it is probable that a combination of genetic , biological , and clinical variables will be required to develop accurate and robust predictive algorithms with which to predict response to drug treatment of jia , and these may need to be disease - subtype specific .
clinical remission in jia can be reached with the use of medication , and is ideally maintained after treatment is stopped   ( criteria for clinical remission on and off treatment are described by wallace et al . ) . however , after discontinuation of mtx treatment , approximately 3050  % of patients relapse [ 2729 ] , suggesting the presence of subclinical disease activity .
this subclinical disease activity is not detectable using clinical and standard laboratory tests , and these patients will not reach stable remission off medication [ 37 ] .
subclinical disease activity makes it difficult to determine when jia patients have reached stable remission and treatment can be stopped .
therefore , a biomarker identifying patients at risk of disease relapse on the basis of the inflammatory status of their disease would be extremely valuable for improving paediatric medical care .
it has been suggested that continuation of mtx treatment for a longer period of time after clinical remission has been reached might reduce the risk of relapse after stopping treatment .
therefore , a randomised clinical trial was conducted to investigate whether longer treatment with mtx , for those who achieved clinical remission , could reduce relapse after mtx withdrawal , and which biomarkers could identify patients at higher risk of relapse after withdrawal .
patients were continuously treated with mtx for either six or 12 months after clinical remission was reached , and then followed after withdrawal of mtx .
however , there was no significant difference in relapse between the two groups [ 37 ] .
on the basis of previously published results from this group , which revealed that mrp8/14 is a marker for subclinical disease activity [ 38 , 39 ] , they determined mrp8/14 levels in serum in both groups .
interestingly , patients with a high relapse rate after discontinuation of mtx had higher levels of mrp8/14 before stopping mtx than patients who did not flare .
thus , serum mrp8/14 levels could be a potential biomarker for predicting which patients will achieve stable remission after withdrawal of mtx , on the basis of inflammatory disease status [ 37 ] .
similar results regarding the potential of mrp8/14 as a biomarker to identify patients at risk of relapse were described specifically for sjia patients .
patients with new onset of active disease or with relapse had higher levels of serum mrp8/14 , compared with patients who had reached stable remission . measurements of mrp8/14 levels at time of stopping treatment revealed that , within up to six months , mrp8/14 levels were higher in patients with disease relapse than patients with no disease relapse .
prediction of patients at risk of relapse was highly accurate , with a sensitivity of 92  % and specificity of 88  % using mrp8/14 levels > 740  ng  ml as a cut - off .
another study investigated whether s100a12 and high sensitivity ( hs ) crp could identify patients who have reached clinical remission on treatment , and are at risk of disease relapse after withdrawal of medication .
high s100a12 and mrp8/14 levels were observed in patients who suffered disease relapse within six months of discontinuation of treatment .
hscrp levels did not differ between patients with disease relapse and those in remission , but this could be caused by the use of inclusion criteria based on normal hscrp levels , because patients with high hscrp levels are more prone to disease relapse .
the patient group which relapsed within three months of stopping treatment had higher median levels of s100a12 and mrp8/14 compared with the group which relapsed later .
s100a12 , mrp8/14 , and hscrp were separately tested for their performance as biomarkers , and s100a12 was the best single biomarker for predicting disease relapse .
treatment strategies for jia have the objective of full clinical remission , but only a very small number of patients remain in clinical remission , off medication , for long periods of time .
a recent study compared the transcriptional profiles of patients with active disease or those in clinical remission on medication ( achieved by treatment with mtx or mtx plus tnf blockade ) with the profiles of healthy children .
differences in transcriptional profile were observed between patients with active disease and those in clinical remission on medication , and also between those in remission off treatment and healthy children .
interestingly , the same group also demonstrated differences in transcriptional profile between patients in clinical remission induced by treatment with mtx or with mtx plus tnf inhibitor , and healthy individuals .
this study confirmed previous findings from the group that the inflammatory status of patients in remission does not return to  normal  .
network analysis suggested a function for hepatocyte nuclear factor 4 alpha ( hnf4 ) , which is expressed by t cells and granulocytes , in controlling genes associated with remission .
such tests need to be reliable , simple to perform , economically feasible , and robust . in addition , they need to be tested and validated for large cohorts of children with jia . where proved reliable
, biomarkers may assist with predicting disease type , course or severity , with predicting response to medication and therefore aiding treatment choices , and with accurate identification of children who can safely stop medication once apparent clinical remission is reached .
the accurate choice of medicines most likely to work for each child , to enable the achievement of rapid remission for all , while avoiding unnecessary exposure to toxic drugs for those who do not need them and reducing unopposed inflammation to a minimum , is now the ultimate objective of modern management of arthritis . to move rapidly towards such ambitious objectives ,
multi - centre and international collaborations are needed to give every child with jia the opportunity to take part in biomarker and cohort studies .
 use of biomarkers in clinical practice has proved extremely valuable and is a rapidly expanding field . however , despite the huge potential of biomarkers , for juvenile idiopathic arthritis ( jia ) there are currently no validated paediatric biomarkers available to help with setting up a more tailored approach on which drug choice could be based , to achieve remission early in the course of disease . 
 early remission reduces burden of disease , limits side effects from toxic and unnecessary medication , and , most importantly , enhances quality of life . 
 several studies have suggested promising biomarkers : these may be a protein , cellular component , mrna , or genetic component , for example a single nucleotide polymorphism ( snp ) . 
 here we describe recent developments in the use of biomarkers for jia and their potential to assist in management of disease by predicting disease phenotype , severity , progression , and response to treatment , and determining when patients have reached stable remission and can safely discontinue treatment . 