Biochemical markers of ongoing joint damage in rheumatoid arthritis - current and future applications, limitations and opportunities

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease associated with potentially debilitating joint inflammation, as well as altered skeletal bone metabolism and co-morbid conditions. Early diagnosis and aggressive treatment to control disease activity offers the highest likelihood of preserving function and preventing disability. Joint inflammation is characterized by synovitis, osteitis, and/or peri-articular osteopenia, often accompanied by development of subchondral bone erosions, as well as progressive joint space narrowing. Biochemical markers of joint cartilage and bone degradation may enable timely detection and assessment of ongoing joint damage, and their use in facilitating treatment strategies is under investigation. Early detection of joint damage may be assisted by the characterization of biochemical markers that identify patients whose joint damage is progressing rapidly and who are thus most in need of aggressive treatment, and that, alone or in combination, identify those individuals who are likely to respond best to a potential treatment, both in terms of limiting joint damage and relieving symptoms. The aims of this review are to describe currently available biochemical markers of joint metabolism in relation to the pathobiology of joint damage and systemic bone loss in RA; to assess the limitations of, and need for additional, novel biochemical markers in RA and other rheumatic diseases, and the strategies used for assay development; and to examine the feasibility of advancement of personalized health care using biochemical markers to select therapeutic agents to which a patient is most likely to respond.


Introduction
It is now widely acknowledged that early diagnosis of rheumatoid arthritis (RA) and aggressive treatment to control disease activity off er the highest likelihood of preserving function and preventing disability. RA is a chronic autoimmune disease characterized by polyarticular infl ammation associated with synovitis, osteitis, and peri-articular osteopenia, often associated with erosion of subchondral bone and progressive joint space narrowing [1]. Th ese features commonly lead to progressive joint damage, impaired function, and progressive disability [2][3][4]. Since roughly half of RA patients suff er disability within 10 years of diagnosis, it is critical to eff ectively treat the disease early to suppress infl ammation and prevent destruction of bone and joint cartilage [5,6]. Treatment is commonly determined by the extent or severity of disease activity, assessed by counting the number of swollen and tender joints, measuring patient-reported outcomes (for example, patient global quality of life assessment), and assaying acute phase responses, such as the erythrocyte sedi mentation rate (ESR) and C-reactive protein (CRP) levels.
While infl ammation markers are clinically relevant, markers that reliably detect ongoing bone and cartilage damage are potentially more useful for timely monitoring of effi cacy of treatment. Joint infl ammation and damage are so far assessed by various imaging methods, including hand and feet radiographs, hand magnetic resonance imaging (MRI), and high-resolution ultrasound of specifi c joints [7]. Biochemical markers of bone and cartilage turnover are also receiving increasing attention in other conditions characterized by joint and/or skeletal infl ammation and damage [8]. Th ey may provide an additional and potentially more sensitive method of detection of active bone and cartilage degradation that is likely to lead to structural damage in RA [0]. An evolving line of evidence suggests that markers associated with clinical response may not be the same biomarkers that predict risk of further joint damage, as verifi ed by radiological progression, and thus diff erent marker combinations are likely to be needed, with specifi c combinations selected for specifi c uses, potentially contri buting to personalized health care [10][11][12]. Prog nostic markers could be divided into at least two categories: those that predict clinical response in terms of signs and symptoms of RA, and those that predict and monitor joint damage, as detected cumulatively by various imaging modalities, and ultimately demonstrated by the clinical manifestations of deformity and dys func tion.
Th e aims of this review are to describe pathobiology that generates biochemical markers of joint metabolism/ damage in RA, including application in assay development; to survey the current use of biochemical markers of joint damage in RA and some other relevant diseases; to discuss the limitations of some of these established biochemical markers, including the need for further research into serum and urine markers, to encourage optimal study designs and sample acquisition; to describe how biochemical markers may allow for diagnosis of patients who are experiencing joint damage with rapid degradation of bone and/or cartilage and thus are most in need of timely, aggressive treatment; and to discuss how advances in personalized health care, including mapping of a patient's specifi c biomarker and clinical profi le, will allow treatment selection according to those that will be most likely to benefi t.

Pathobiological processes associated with progression of joint damage, and biochemical markers of joint damage
Th e diff erent cellular phenotypes involved in joints (osteoblasts, osteoclasts, chondrocytes, macrophages, B cells, T cells, fi brobast-like synoviocytes and macro phages) play distinct complex and inter-related roles in the pathogenesis and progression of RA joint damage [13]. Subchondral bone erosion, sclerosis and articular cartilage degradation leading to joint space narrowing are central features of joint damage in RA. Synovitis and osteitis associated with osteoclast activation and degra da tion of bone by matrix metalloproteinases (MMPs) and cathep sin K appear to precede erosions visualized by MRI or radiography [13][14][15][16][17]. Further, cytokines such as IL-1, TNFalpha, IL-6, and IL-17 stimulate chondrocyte activa tion and expression of MMPs and aggrecanases, resulting in articular cartilage degradation. Th us, a wide range of processes contribute to the pathobiology of joint damage that eventually leads to joint failure [3,10,11,14] (Figure 1). A detailed discussion of the cellular inter actions and molecular pathways involved in bone and cartilage damage in RA is out of the scope of this review, and has been documented elsewhere [3,10,12-15,17,].
Th e generation of a tissue-specifi c biochemical marker is presented in Figure 2. Th e enzymes in the infl amed joint generate specifi c biochemical metabolic products from the extracellular matrix; the actual protein frag ments of type II collagen and aggrecan that are the result of pathobiological actions in the joint are schematically presented in Figure 3. Th ese specifi c products, which will be described, can be measured [7,18], facilitating assess ment of various molecular, cellular and pathophysio logical processes in the joint. Each marker may provide unique insights into the pathology of the disease by allowing quantitative information on the level of disease activity in terms of target tissue damage, on the action of cytokines driving disease progression, and on the specifi c mode of action and potential effi cacy of therapeutic interventions. Th ese features provide perspective for the characterization of the ongoing pathobiology using sets of biomarkers that potentially describe the type of damage occurring. A combination of specifi c biomarkers may thus provide more detailed and accurate information on joint pathology and ongoing structural damage than individual markers.
As described above, the biochemical markers may be useful by providing quantitative information on the pathology and unique processes associated with joint damage in RA. In addition, from a patient-management perspective, the biochemical markers may be useful for the diagnosis of patients with ongoing, active damage to, and degradation of, bone and/or cartilage, for early detection and monitoring of response to treatment, and for personal izing health care. Patients with such ongoing, active damage and degradation of bone and/or cartilage might be classifi ed as 'rapid progressors' and are those most in need of eff ective treatment. Th ey may be identifi ed by detection of abnormal serum and/or urine levels of bone, synovium and/or cartilage degradation/turnover markers, prior to established, irreversible damage being identifi ed using one or more imaging modalities. Early detection and monitor ing of response to treatment potentially provides more rapid verifi cation of control of joint damage than improve ment in clinical symptoms or imaging changes, since a minimum of 6 months is needed to ascertain radiological progression, although newer MRI technologies may detect changes in osteitis and synovitis within several months. Health care can be personalized by identifying patients most likely to respond or not to a particular treatment, thus enabling informed selection of an appropriate thera peutic agent, as well as timely verifi cation of its expected effi cacy.

Biochemical markers as predictors of progression of structural damage
Biochemical markers of bone turnover have been used as standard practice to measure the eff ects of therapy in osteoporosis (OP), a slowly progressing condition [17]. For example, early changes in CTX-I (C-terminal telopeptide of collagen type I), a marker of bone resorption, and changes in osteocalcin, a marker of bone formation, can be used to predict increases in bone mineral density [8]. In contrast to imaging techniques, biochemical markers of bone and cartilage turnover, measured in serum or urine samples collected during fasting or as second morning void specimens, show clinically relevant changes over a larger range compared with the imprecision of the assay (8% to 10%) [17]. A typical decrease of 50 to 80% or an increase of 100 to 200% is observed in the level of biochemical markers within days to weeks after initiation of treatment with anti-resorptive or anabolic drugs [17]. However, the respective change in bone mass ranges from 6 to 7% after 2 years of bisphosphonate therapy, which is a comparatively small increment relative to a precision error of 1 to 2% for bone mineral density (BMD) measurements, as reviewed recently [17], and thus could be considered inferior to the dynamic range observed with biochemical markers. Because biochemical markers are sensitive and dynamic indicators of tissue turnover, they have the potential to provide information on treatment effi cacy more rapidly than a variety of imaging methods ( Figure 4) [16]. Th is use of biochemical markers of bone turnover has so far been validated in OP, as have markers of cartilage turnover in osteoarthritis (OA) [8,17]. In OP, a dynamic biochemical marker such as CTX-I changes within days of initiating treatment with anti-resorptives or the anti-receptor activator of NF-kB ligand (RANKL) drug denosumab, whereas BMD im prove ments can only be reliably detected over 6 to 12 months. Similarly, urinary CTX-II (C-terminal telo peptide of collagen type II) levels have been shown to predict articular cartilage degradation [19] in OA. Th e same markers have been examined in RA

cells, T cell subsets (including regulatory T cells)
, and fi brobast-like synoviocytes, each playing distinct complex and interrelated roles in its pathogenesis and progression. This cellular diversity highlights the need for biomarkers for a range of pathological events. Diff erent markers of cell signaling (for example, receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG)), cell diff erentiation, collagen I and II degradation and turnover, matrix production, and matrix degradation and the enzymes mediating that degradation may be measured. The pleiotrophic cytokines IL-1β, TNF-α, IL-6, and IL-17, as well as several other cytokines and chemokines, are associated with the induction of matrix metalloproteinases (MMPs), as well as osteoclast diff erentiation, activation and release of cathepsin K [36]. This range of interactive events leads to progressive joint destruction if not managed attentively, for example, using tight control strategies [15,18,22,104,140,141]. C2C, type II collagen fragment; CIIM, MMP mediated type II collagen degradation; CTX-I, C-terminal telopeptide of collagen type I; CTX-II, C-terminal telopeptide of collagen type II. [20]. CTX-II as a marker of cartilage (collagen II) degradation and CTX-I as a marker of bone (collagen I) degradation in RA at 4 and 12 weeks have been demonstrated to predict joint damage (Tables 1, 2 and 3).
Research eff orts are underway to apply these principles to proactive management of RA to enhance the detection and prevention of joint damage. X-ray imaging is the standard technique for diagnosis and measurement of effi cacy of therapies aimed at inhibiting joint damage. Further eff orts are ongoing to validate the use of MRI in this process, and even combine the use of biochemical markers and imaging modalities [7,21]. In RA, joint damage characterized by subchondral bone erosions and joint space narrowing, rather than BMD as in OP, is measured by various scoring methods applied to X-rays of hands and feet. However, X-ray imaging in both diseases is encumbered by rather low precision and could conceivably benefi t from combination with biochemical marker analysis ( Figure 4).

Need for biochemical markers to facilitate treatment decisions
Recently, three biological agents with novel mechanisms of action, rituximab, abatacept and tocilizumab, have become available for the treatment of RA, adding to the armamentarium already containing the approved TNF-α inhibitors (infl iximab, etanercept, adalimumab, certoli zu mab and golimumab). Clinical studies with these agents have demonstrated that they are eff ective in RA patients who did not respond to treatment with at least one disease-modifying antirheumatic drug (DMARD) and/or TNF inhibitor. In the absence of head-to-head trials, the use of specifi c biochemical markers may aid in diff erentiating the onset and/or the magnitude and even duration of effi cacy of the diff erent drugs, and in understanding which patient may respond best to a given intervention. Th e early identifi cation of responders and non-responders to the increasing range of treatments for RA, a disease recognized to lead to loss of function and disability if not aggressively treated, will prove valuable to patients, regulators, healthcare providers and payers. Th e emphasis in RA management today is on early diagnosis and treatment to prevent the progressive joint deteri oration predominantly driven by infl ammation [22][23][24]. Selecting the most appropriate intervention has become increasingly complex because, for example, combinations of some therapies have proven more eff ective in clinical trials than single agents alone and also because diff erent interventions may be more appropriate than others according to the stage and risk of disease progression in individual patients. In some patients, joint damage progresses slowly over time and then begins to progress in a more rapid and dynamic fashion. In those where infl ammation is more severe, structural damage can occur ). The most abundant cartilage proteins are collagen type II and aggrecan. Protease-generated fragments of collagen type II and aggrecan produced through the action of these important enzymes, which may be relevant molecules in tissue destruction, can be used to monitor tissue turnover. These fragments, such as C-terminal telopeptide of type II collagen (CTX-II), may be used in clinical settings, in preclinical models and in simple ex vivo and in vitro systems. Figure adapted with permission from [8].
within just a few months after disease onset. Consequently, the greatest opportunity to change the course of the disease could be through the identifi cation of those patients who either have, or are at risk of developing, rapidly progressive disease. Using biomarkers to predict risk and response to therapy will not only aid the selection of an appropriate, eff ective intervention for the individual but will also protect patients with less severe disease from possible aggressive over-treatment and toxicities, and may have a signifi cant infl uence on allocation of health care resources. Several biological markers and clinical indicators have been discovered to identify such patients.

Biochemical markers of joint damage
Currently, there is no single clinical or laboratory characteristic that identifi es RA patients with rapidly progressing joint damage and systemic bone eff ects. Th e best-characterized predictors of risk for rapid progression are the number of swollen joints and levels of acutephase reactants such as CRP and ESR. Th is is not surprising because swollen joints are a clinical manifestation of synovitis, and the acute-phase response acts as a biomarker of pro-infl ammatory cytokine production. It is well documented that elevated CRP is associated with increased risk of radiological progression in RA [24,25], and correlation between synovitis and subchondral bone The amino-and carboxy-terminal pro-peptides PINP (amino terminus propeptide of type I procollagen), PICP (carboxyl terminus propeptide of type I procollagen), PIINP (amino terminus propeptide of type II procollagen) and PIICP (carboxyl terminus propeptide of type II procollagen) in collagen type I (a) and collagen type II (b) are used to defi ne protein formation, as they are released during formation of the matrix. (a) In contrast, the degradation markers ICTP (type I collagen; MMP mediated) and C-terminal telopeptide of type I collagen (CTX-I; cathepsin-K mediated) located in the carboxy-terminal telopeptide are found in body fl uids after degradation of collagen type I. (b) The CTX-II (MMP mediated) degradation marker is located in the carboxy-terminal telopeptide in collagen type II. Coll 2-1, TIINE, C2C, and C2-3/4C are degradation markers located in the helix of collagen type II. (c) The aggrecan molecule is shown with the MMP cleavage sites (upward arrows) and ADAM-TS (a disintegrin and metalloproteinase with thrombospondin motifs) cleavage sites (downward arrows). CIIM is a novel MMP mediated type II collagen degradation marker [142].   erosions has been established [26][27][28][29]. While not all patients with high disease activity that manifests in high swollen joint counts and elevated CRP are immediately eligible for biologic therapy, those who also show ongoing degradation of joint structure proteins may benefi t from the most intensive therapy, especially if effi cacy can be detected early to manage benefi t and risk considerations [25]. Th e focus of research into joint damage biomarkers has been the identifi cation of proteins that might be surrogates of whole tissue metabolism and of bone and cartilage loss. One approach to identifying pathologically relevant molecules is to combine tissue-specifi c protein markers with the pathological expression of proteolytic enzymes. Th e action of enzymes on extracellular matrix components results in matrix degradation fragments, or neoepitopes. Th e most abundant molecules in the articular cartilage extracellular matrix are collagen type II and aggrecan. Th ese proteins are sequentially degraded when cartilage damage occurs in either RA or OA. Proteasegenerated fragments of collagen type II and aggrecan produced by MMPs and aggrecanases (ADAM-TS) are considered relevant molecules in cartilage degra da tion [8] (Figure 3). Whole joint tissue pathophysiology may be assessed by the one or more markers of cartilage degradation, but these are only a subset of a larger panel of markers that provide information on bone and infl amed synovial tissue in the joint ( Table 1). As also described in Table 1, additional cartilage degrada tion markers are becoming available, aimed at more accurate and precise detection of articular cartilage damage. Specifi c fragments of cartilage proteins have been identifi ed as specifi c markers of joint damage. Much of this work has been applied according to the US Food and Drug Administration critical path for the development of biochemical markers in translational research [8], where such markers may be applied in both preclinical and clinical research settings.

Joint turnover markers
Infl ammatory joint diseases such as RA lead to alterations in the metabolism of the articular cartilage and subchondral as well as periarticular bone [30][31][32][33][34][35]. Unique markers have been developed, and others are under development, to refl ect diff erent pathobiologic processes. How these processes occur at diff erent stages in the patho genesis, and result in unique metabolic products of joint infl ammation, is discussed in the sections below.

Cartilage turnover markers
Cartilage turnover normally occurs in a controlled fashion, with a balance between degradation and formation. However, in the infl amed joint, an imbalance is skewed towards degradation rather than formation [36]. Formation and degradation can be monitored by measuring several unique molecules generated during cartilage degradation and turnover [17]. Cartilage is predomi nantly composed of collagen type II (comprising 60 to 70% of the dry weight of cartilage) and proteoglycans (10% of dry weight), of which aggrecan is the most abundant [37]. Th e key mediators of cartilage degradation include the MMPs and the closely related aggrecanases, which are members of the ADAM-TS family [38,39].  Aggrecan is degraded by both MMPs and aggrecanases, whereas collagen type II is degraded by MMPs [40]. Th e action of these proteases results in the release of collagen and aggrecan peptide fragments that can be measured by ELISA-type assays both in vitro and ex vivo [17] ( Figure 4). Since collagen type II is the most abundant protein in cartilage, several diff erent degradation fragments of collagen type II have been identifi ed as useful for monitoring the impact of joint infl ammation on cartilage [17,41]. One example of a novel biochemical marker based on neoepitopes [16] is CTX-II, an MMP-generated neo epitope derived from the carboxy-terminal part of type II collagen [42,43]. Measurement of CTX-II has proven useful for monitoring degradation of type II collagen in experimental models assessing cartilage degradation [17,42,43]. Cartilage degradation and formation can be effi ciently studied in ex vivo cultured explants of bovine articular cartilage [40,[44][45][46][47]. In this model, a high rate of cartilage degradation can be induced, for example, by the combination of TNF-α and oncostatin M, which induce cartilage degradation in a time-and concentrationdependent manner. Th e role of MMPs is demonstrated by the abrogation of cytokine-induced CTX-II release by the addition of the MMP inhibitor GM6001, but not the cysteine proteinase inhibitor E64. Further, biochemical studies showed that both MMP-9 and MMP-13 had the ability to generate CTX-II fragments [40]. In addition, immunohistochemical localization of CTX-II revealed that it is highly present in areas corresponding to proteoglycan depletion in TNF-α-and oncostatin Mtreated explants [40]. Additional analysis of CTX-II demon strated that it was localized in the damaged areas of the articular cartilage [48][49][50]. In clinical studies, high levels of CTX-II have been shown to be associated with the diagnosis of OA and to predict progression of RA and OA joint damage [51]. Th us, the assay for this MMPgenerated collagen type II neoepitope, CTX-II, is an example of a clinically and pathologically validated indicator of cartilage degradation, although its responsiveness to therapeutic intervention continues to undergo intensive investigation. With further charac ter i zation in prospective clinical trials, the CTX-II assay may provide an example that assays for neoepitopes generated by a specifi c combination of enzyme and matrix molecules are potentially relevant for monitoring risk of joint damage and impact of therapy. Th e development of assays to assess cartilage degradation and formation is not limited to just CTX-II (Table 4). Degradation markers include urinary TIINE, serum C2C, C1C2, Coll-2-1, ICTP and HELIX-II, and synthesis markers include PIINP and PIIANP, as they are based on propeptides. COMP and YKL-40 have also been used to assess cartilage degradation, but have also been characterized to detect matrix turnover [17,19,45,47,.

Bone turnover markers
Bone turnover is a continuous process that ensures calcium homeostasis and bone quality [75]. Th e total skeleton is completely replaced every 10 years on average, Cat K Osteoclasts Osteoclast number [110] emphasizing the dynamic nature of this organ and refl ecting changes in endocrine function as well as the eff ects of disease, drugs, and nutritional defi ciencies [76]. Perturbation of this delicate balance leads to pathological conditions such as OP and fracture risk, that is, bone loss. Bone turnover is mediated by activated osteoclasts, which degrade the established bone matrix, and osteo blasts, which form new bone matrix, two processes that, under normal circumstances, are tightly coupled and balanced [77]. Th e primary osteoclast driver is RANKL [78], although co-stimulators such as the cytokines IL-1β, IL-6 and/or TNF-α co-stimulate osteoclasts to secrete cathepsin K into the resorption lacunae [79,80], resulting in degradation of the organic matrix of bone. Type I collagen is the most abundant protein in bone [75], and its degradation by cathepsin K leads to the release of the CTX-I or N-terminal telopeptide of collagen type I (NTX) neoepitope [81,82] ( Figure 5). CTX-I levels increase in line with elevated levels of IL-6 after the menopause, indicating increased osteoclast activity and bone resorption [83,84]. CTX-I can be measured in both urine and serum and decreases rapidly in response to anti-resorp tive treatment in OP [84][85][86]. Decreased CTX-I levels within 4 weeks of initiating anti-resorptive therapy corre late with BMD increase at 1 year, demonstrating the effi cacy of the intervention [87][88][89]. As a result, CTX-I is being used in a large number of studies [88][89][90][91][92][93] to monitor the effi cacy of anti-resorptive therapies.
In RA a variety of factors, such as the impact of systemic infl ammation, corticosteroid use, and menopause, may infl uence bone resorption, bone turnover and skeletal status over time. Activated osteoclasts participate in altered bone balance since absence of osteoclasts or absence of osteoclast activities will lead to attenuation of bone resorption but only modest eff ects on cartilage degradation [3,94,95]. Th e role of cathepsin K has been extensively studied, and the data are somewhat confl icting for RA [40,[96][97][98]. Levels of cathepsin K are in creased in RA, indicating that it can be used as a marker [99,100], although cathepsin K does not appear to be the primary enzyme driving bone destruction in RA [98,101,102]. CTX-I levels correlate only to some extent with joint damage in RA, and are likely also infl uenced by loss of skeletal structure/osteopenia/OP, which are also prevalent in RA [22,103,104]. MMPs also play a role in infl ammation-associated bone loss [105,106]. Studies showing that the MMP-derived collagen type I fragment ICTP is increased in RA may indicate that osteoclasts induce MMP-mediated matrix degradation under these circumstances [82,107,108]. Infl iximab and tocilizumab treatment have been shown to reduce ICTP levels, as well as osteoclast numbers [103,109], consistent with osteoclast MMP-mediated bone degradation in RA. However, a direct link between the production of ICTP and osteoclasts has not been demonstrated yet.
As illustrated in Figure 5, a range of diff erent markers is available for assessing bone balance in RA. Th e most  Compilation of parameters known to infl uence biological variation or analytic performance of a given biochemical marker. These parameters include, but are not limited to, biological variation or analytical performance of a given biochemical marker. a Age, gender, menopause status, ethnicity, duration of rheumatoid arthritis, prior treatments such as TNF antagonists, concomitant medications such as corticosteroids, estrogen, SERMs, and bispohosphonates, and comorbidities such as osteoporosis, diabetes, and hypertension with or without renal insuffi ciency.
important markers are those of bone formation (PINP, osteocalcin, bone specifi c alkaline phosphatase (BSAP)) and bone resorption (for example, NTX and CTX), while assessment of osteoclast numbers by levels of the enzymes TRACP 5b and cathepsin K has more recently provided additional information complementary to bone resorption markers [74,110]. Another TRACP isoform, TRACP 5a, is a macrophage marker, and appears to corre late with infl ammation [111]. Circulating levels of the formation marker PINP correlate with histomorphometric indices of bone formation [17]. Osteocalcin levels are characteristically low in RA, associated not only with systemic bone loss but also corticosteroid treatment, and levels may stabilize or increase with eff ective control of infl ammation [112][113][114].
In conclusion, the infl amed joint is composed of several tissues, each of which is subject to degradation and dysregulated collagen and matrix metabolism, in contrast to a normal joint where the balance between formation and degradation is tightly controlled. Changes in biochemical markers generated as a result of dysregulated metabolism may be useful for timely detection of changes in response to treatment in order to limit joint damage and bone loss in RA.

Currently available biochemical markers of joint damage
Th e strategy for developing biochemical marker assays has evolved with experience in applying results from disease diagnosis and prognosis as well as from monitor ing the eff ects of treatments for conditions commonly associated with joint damage. Th e selection of particular assays depends on the objectives for study, but in most settings these include: examination of the mechanism of action to verify potential benefi ts in limiting joint damage; prediction of risk of joint damage; diagnosis of ongoing bone and/or cartilage degradation in joints; and monitoring for timely detection of onset of action and maintenance of eff ect to limit joint damage.
Currently available and commonly used biochemical marker assays are described in Table 1. Th is is not intended to be an exhaustive list; rather, it is intended to orient the reader to assays that have been commonly reported in clinical studies in arthritis, together with several assays that are currently being examined for improvements to meet the above objectives.

Biochemical markers in ankylosing spondylitis -examining unique features of dysregulated bone and cartilage metabolism
Due to the paucity of information provided by standard clinical and laboratory parameters to guide treatment decisions, several of the biomarkers studied in RA have been analyzed in other infl ammatory joint diseases, particularly spondyloarthritis (SpA), on the basis that these disorders may share aspects of pathophysiology with RA. Th ere has been particular interest in evaluating biomarkers in AS that refl ect disease activity and predict Figure 5. In bone, cell activation, cell diff erentiation, matrix production, matrix degradation and the enzymes mediating that degradation may be measured by diff erent markers. Each marker provides unique information and may indicate both pathological aspects and serve as a surrogate measure of the mode of action and potential effi cacy of therapeutic interventions [85]. BSAP, bone specifi c alkaline phosphatase; CTX, C-terminal telopeptide of collagen; ICTP, collagen type I fragment; NTX, N-terminal telopeptide of collagen type I; OC, osteocalcin; OPG, osteoprotegerin; PICP, carboxyl terminus propeptide of type I procollagen; PINP, amino terminus propeptide of type I procollagen; RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB ligand. structural progression [13,15,21,115]. For disease activity, CRP and ESR lack the sensitivity seen in RA, as these markers are elevated in only about 50% of ankylosing spondylitis (AS) patients [116]. Unlike RA, they also correlate poorly with clinical measures of disease activity, although good correlations have been noted with MRI evidence of infl ammation in the spine [117,118]. In contrast to RA, they do not appear to predict progression of structural damage, although similar to RA, CRP does predict clinical response to anti-TNF therapy [119,120].
Th e primary biomarker refl ecting tissue turnover related to infl ammation in AS that has been analyzed is MMP3. Most studies have shown lower levels of MMP3 in SpA than in RA. Levels are elevated mainly in patients with concomitant peripheral joint infl ammation compared to those with only axial infl ammation, and levels correlate with the histopathological grade of infl ammation [121,122]. As for RA, there is evidence that levels of MMP3 can predict progression of radiographic changes, although for AS this means new bone formation rather than the erosive changes documented in RA [123]. Th is fi nding is one observation that supports the concept of a link between infl ammation and ankylosis in AS. Reductions in MMP3 levels following anti-TNF therapy correlated with reductions in CRP, although MMP3 levels have not been shown to predict clinical response [124].
Biomarkers refl ecting cartilage turnover have been analyzed in limited cross-sectional studies of patients with AS. Elevated levels of CPII and the aggrecan 846 epitope were observed, as they were in RA [125], and normalization was seen with anti-TNF therapy [126]. One study has shown that urinary CTX-II may predict progression of structural damage in AS, as also documented for RA [127]. However, unlike RA, the collagen II degradation markers C2C and C1-2C were not elevated.
Assessment of biomarkers refl ecting bone turnover in SpA have shown variable results depending on the stage and activity of disease, but most studies have reported lower levels for markers of bone resorption than in RA [128]. A major inhibitor of osteoblastogenesis, DKK-1, is markedly elevated in RA but is not predictive in AS [125], while sclerostin is increased in RA and reduced in AS [126]. Th ese changes are consistent with the excess bone formation observed in AS and impaired bone formation in RA.
In the current context, this use of biochemical markers in AS emphasizes that biochemical markers of bone and cartilage may be applied to SpA in well-controlled settings and studies.

Major clinical fi ndings with selected interventions and cohort studies
Bone and cartilage biomarkers have been used with various levels of success in both degenerative and infl am matory joint disease. Table 2 shows those applicable to RA in combination with X-ray imaging and Table 3 provides the current available publications on MRI and biochemical markers in RA. Th ese tables clearly indicate that a subset of markers have already proven useful for investigating effi cacy in RA, although surprisingly few combinations of MRI and biochemical markers are currently used. Th ese tables also do not constitute a full list of relevant studies; important information is available in other publications to complement the condensed infor mation here [19,20,22,51,104,123,126,127,[129][130][131][132][133][134][135][136][137].

Strategies for use of biochemical markers to enhance the benefi t:risk ratio of RA therapies
Th e lack of consensus on the optimal biochemical marker combination in RA is understandable given the varying outcomes from diverse studies in which their predictive value has been assessed. However, these diff ering outcomes are likely due, at least in part, to diff erences in patient populations, such as varying duration of RA, and confounders, such as current and prior treatments, concomitant corticosteroids and other medications, as well as comorbid conditions (Table 4). Typically, studies with biologics with novel and unique mechanisms of action often recruit patients who have failed to respond to one or more therapies and were receiving a variety of concomitant medications. Th us, even though patient populations at fi rst glance may seem somewhat similar, important diff erences exist and these need to be carefully considered when interpreting results.
Based on our current knowledge on RA, diff erent marker combinations may be useful at diff erent disease stages for identifying severity and risk of progression of joint damage. Th is concept is illustrated in Figure 6a, and elaborated in Figure 6b-d. However, the use of biochemical marker profi les to identify individual patients who will respond to a particular intervention, or are more likely to experience rapid progression of joint damage, still remains a major challenge.
Th e pathology of RA appears to consist of a variety of diff erent phenotypes. If RA is left un-segmented and the population treated as a whole, the proportion of patients experiencing remission is relatively low in most clinical trials. As illustrated in Figure 6b, if a biomarker combination can identify a subset of patients representing a given phenotype who will respond to treatment, or demonstrate a superior response to a specifi c therapeutic intervention, then response rates in this patient subset will be far greater than those in the unstratifi ed population. Th is is an important socio-economic opportunity. By targeting the optimal treatment to patients who will derive the most benefi t, the most favorable benefi t:risk ratio will be obtained.
Th e optimal biomarker combinations for specifi c purposes and questions need to be carefully investigated, as illustrated in the fi gures in this paper. Combinations may depend on the duration and stage of disease in addition to the disease activity and associated eff ects on bone and cartilage tissues. It is now recognized that anti-TNF therapies may limit joint damage, even in clinical nonresponders, and responders to DMARD treatment may continue to experience ongoing joint damage, albeit at a slower rate [23,138]. Th us, a specifi c combination of biochemical markers may not enable discrimination between clinical responders and non-responders for both radiological progression and patient assessment schemes as outlined by the American College of Rheumatology responder criteria or Disease Activity Score systems. Th is feature of current therapies remains a further challenge for the use of 'optimal' combinations of biochemical markers and highlights the potential usefulness of biochemical markers of active joint damage.
Lastly, as illustrated in Figure 6c,d and as discussed for the Burden of Disease, Investigative, Prognosis, Effi cacy of Intervention and Diagnostic (BIPED) categorization of biochemical markers [139], diff erent questions can be addressed by using these tools. As illustrated in Figure 6c, prognostic markers are those able to predict who will progress most rapidly. Th is is an important part in identifi cation of those in most need of treatment. Th e prognostic marker may also allow for identifi cation of particular patient phenotypes that will respond to treatment (Figure 6a,b). A marker of effi cacy as illustrated in Figure 6d is a measurement at baseline or a temporal measurement compared to baseline, allowing the Rheumatoid arthritis (RA) may consist of many diff erent subphenotypes, with similarities and dissimilarities, as illustrated by the overlap and non-overlap of the diff erent colored circles. If this population is left unsegmented, and the population treated as a whole, a relatively low number of responders may be identifi ed. (b) A biomarker combination may identify a subset of patients representing a given phenotype that will respond to treatment, or respond preferentially to a particular therapeutic intervention, increasing overall response rates. (c,d) Diff erent questions can be addressed by the use of biochemical markers. Each may require a diff erent biomarker subset. (c) Prognostic markers are those able to predict which patients will progress most rapidly. This is important for identifying those patients most in need of treatment. (d) A marker of effi cacy will allow interpretation of potential effi cacy far earlier than traditional radiological-based changes. interpretation of potential effi cacy ahead of traditional radiological-based techniques, such as illustrated in Figure 2. In particular, in the fi eld of certain bone diseases, CTX-I is a surrogate marker of effi cacy, aiding the prediction of a patient's response to treatment before standard radiological assessment is possible [16].

Biomarker classifi cation
Not all biochemical markers provide the same information. Some may be diagnostic, whereas others may aid prognosis, and others indicate the potential effi cacy of interventions. Th us, one biomarker that may fail in one function or scenario may provide important information in another. Th is highlights the need for a framework to understand terminologies in the development and use of biochemical markers. Th e recently proposed BIPED classifi cation, developed by the Osteoarthritis Biomarkers Network, which is funded by the US National Institutes of Health, has further highlighted the need for understanding biomarkers and their use [139]. Th e BIPED classifi cation provides specifi c biomarker defi nitions with the goal of improving the development and analysis of OA biomarkers and of communicating advances within a common framework. Briefl y, the fi ve defi nitions for OA are burden of disease, investigative, prognostic, effi cacy of intervention, and diagnostic. Burden of disease markers assess the severity or extent of disease, for example, severity within a single joint and/or the number of joints aff ected. Investigative is an investigative marker with insuffi cient information to allow inclusion into one of the existing biomarker categories. Th e investigative category includes markers for which a relationship to various normal and abnormal parameters of cartilage extracellular matrix turnover has not yet been established in human subjects. Th e key feature of a prognostic marker is the ability to predict the future onset of OA among persons without OA at baseline or the progression of OA among those with the disease. An effi cacy of intervention biomarker provides information about the effi cacy of treatment among persons with OA or those at high risk for development of OA. Diagnostic markers are defi ned by the ability to classify individuals as either having or not having a disease.
Th is very simple yet elegant classifi cation could be used in other disease indications, such as RA, to foster optimal use, and avoid miscommunication of the benefi ts of selected biochemical markers.

Confounders that infl uence the application and interpretation of biochemical marker assay results
As many factors aff ect the measurement and inter pretation of changes in levels of biochemical markers, a critical review of sample acquisition, storage and assay parameters must be undertaken to optimally assess the reliability of biochemical marker analysis. Some of these considerations are highlighted in Table 4, and the reader is referred to the referenced papers for an in-depth discus sion of the individual assays and guidance for appropriate, evidence-based interpretation of their results. Multiple biological or analyte-related factors, assay specifi cations, study parameters and the context in which the results are interpreted are often underestimated and ignored in the study design phase but can have tremendous impact on the fi nal interpretation of the results.
Technical performance strategies for reproducible and reliable biochemical marker analysis include, but are not limited to, the following parameters. Th e analytical method must be validated by the laboratory for each biomarker used in a clinical study before the laboratory begins analyzing samples from the study. Although manu facturers' kit inserts provide useful assay parameters, it is mandatory that each laboratory verifi es it can reproduce these parameters. Th e validation should be performed on the same sample matrix (serum, plasma, urine or synovial fl uid) as collected in the clinical study. Results obtained from serum are not necessarily the same as those from plasma, for example. Th e analytical validation should include calibration curves, with at least six non-zero standards, intra-and inter-precisions and accuracy, the range of quantifi cation and sensitivity (lower and upper limits of quantifi cation, limit of detection, specifi city and selectivity, recovery, stability and dilution linearity. Th eoretically, to estimate intraand inter-run accuracy and stability, fi ve diff erent validation samples should be analyzed in duplicate or more in at least six diff erent runs. One of the major problems with assays (especially microtiter plate-based assays) is reagent-lot variation, indicating a lack of assay robustness. Quality control (QC) samples with predefi ned validated ranges must be analyzed together with the calibrators and the study sample in each run. Th ese QC samples must be prepared in the same matrix as the study samples and, whenever possible, must cover the range of the standards curve (lower, middle and upper limits). Th e run must be accepted (or rejected) based on the QC acceptance criteria (typically, a 4-6-X rule, where X is a selected percent deviation from nominal value), but also on the results of the calibration standards (backcalculated value within 20% of nominal). Lastly, whenever possible, batches of samples collected during the fi rst visits of the patients, when changes in biomarker levels in response to drug treatment could be theoretically detected, should be assayed together in the same run. Th is should further minimize inter-assay variation.
Th ese examples serve to highlight that biochemical marker analysis includes a range of parameters that need to be carefully considered and accounted for in optimal assay performance, which eventually will impact the results of the clinical trials.

Conclusion
RA is often characterized by progressive joint damage that, if not arrested by treatment, often leads to substantial limitation of function and progressive disability. It is evident that the nature of progressive joint damage varies considerably, with some RA patients experiencing more rapid progression than others, based on underlying pathobiology, levels of response to treatment, duration and stage of disease, as well as comorbidities and concomitant medications. Patients with rapidly progressing joint damage may particularly benefi t from early aggressive treatment with a biologic agent. Consequently, the identifi cation of patients with ongoing joint damage and assurance that treatment is limiting cartilage degradation and improving bone balance is important in preventing irreversible joint damage. Biological markers and clinical measures can be used to help identify this group of patients, including elevated CRP levels and the number of swollen and tender joints. Additional application of biochemical markers, which are able to sensitively detect ongoing joint damage, may facilitate the appropriate use of targeted therapy in RA and help reduce the progression of joint damage in these patients.

Competing interests
MAK and CC are stockholders of Nordic Bioscience. All others are full time employees of their respective institutions which warrant full disclosure.

Author contributions
MAK and TW wrote the fi rst draft of the manuscript and outlined the paper with AP. KH wrote sections on bone biology. WPM contributed with sections on AS and general pathophysiology. HG provided valuable imaging advice and discussion. PV and TS wrote assay characteristics and challenges sections. GS provided discussion on pathophysiology and helped outline the manuscript. CC and PQ compiled the current biochemical markers section. ACB-J participated in all aspects of this process. All authors approved the fi nal version of the manuscript.