Evolving Biomarkers in Osteoarthritis
Accurate and efficient diagnosis and prognosis are central to effective patient care. Localized pain, discomfort, or an annual check-up is usually the starting point for patient examination by a physician. But the route of patient care taken by a physician depends on several factors derived from blood tests, radiographs, magnetic resonance imaging (MRI) scans, or a physical examination, all of which generate a set of biomarkers advising the physician on the next step in patient care. Thus, conceptually a biomarker is any set of parameters that when out of the normal range would inform the physician of abnormal body physiology or injury in the patient. In the field of osteo-arthritis (OA), however, a physician has very little on which to plan an effective, in-depth patient care stratagem to prevent OA progression. Patients are usually seen when the disease has advanced to a stage at which further progression cannot be prevented. At this stage, a physician′s recommendations are limited to palliative or salvage procedures such as activity modification as in rest and/or joint immobilization, anti-inflammatories, analgesics, or total joint arthroplasty. These limitations in therapeutic options reflect a lack of clinically relevant diagnostic or disease-staging methodologies and the unavailability of any disease-modifying treatments, compounded by an incomplete understanding of OA pathogenesis in conjunction with the heterogeneity of the human population.
Like several other chronic diseases, OA is characterized by a silent phase of the disease when the individual is not aware that the disease process has begun. Fig. 3.1 is a representation of the phases of OA in humans (modified from Kraus 2010)1 where bio-marker validation is desirable for phases 0 and 1 but is currently hypothetical. This has led to a concentrated movement in search of biomarkers to detect early OA. During early OA, though an individual may appear normal on radiographs or MRI scans, abnormalities in joint physiology may indicate OA initiation by elevations in the levels of cartilage breakdown products or markers of inflammation. These biochemical markers are the next wave in OA diagnosis, monitoring OA progression, understanding changes in body physiology, and determining efficacy of pharmaceutical interventions. An OA biochemical biomarker could be any molecule that would act as a surrogate to inform the physician of either disease initiation or progression and allow for accurate disease staging and prognosis for determining optimal preventive and therapeutic strategies. Ideally biomarkers for OA will be joint-specific and detectable during the silent, nonsymptomatic phase of OA. If possible, biomarkers will also be able to distinguish age- and trauma-related changes to allow a physician to assess the degree of damage after injury.
To derive the maximum benefit from a biomarker it would be advisable to arrive at a consensus for a definition of the various stages of the disease process. In OA, often this is lacking. Many individuals are often reported to have radiographic OA as defined by joint space narrowing. However, most of these individuals often do not have symptomatic OA as defined by pain or discomfort. But joint space narrowing would be due to changes in articular cartilage structure that would reflect changes in articular cartilage composition. An individual at such a stage of OA, radiographic but nonsymptomatic, may not be denoted clinically as having OA and therefore would be bypassed for treatment. This puts the burden of proof on the biomarker as it should be validated as truly representative of the disease process, should be an irrefutable proof of disease initiation, and should be universally recognized as such. A late-stage biomarker more representative of disease progression is not likely to be of use in detecting the elusive, early OA phases. Thus, a primary goal of OA bio-marker development is to catch the disease in the early stages to prevent the ravages of late-stage OA. Several biomarkers are currently in use but have varying degrees of acceptance for their utility in OA ( Table 3.1 ). The OA Biomarkers Global Initiative has been organized by Osteoarthritis Research Society International (OARSI) in the recent past to accelerate OA biomarker development.2 A documentation by the OARSI Food and Drug Administration (FDA) initiative provides a summary and guide for application of in vitro, soluble, biochemical biomarkers for monitoring OA and pharmacological trials and also provides a scheme for the classification of these biomarkers.3
Biomarkers Derived from Type II Collagen
The fibrillar type II collagen (Col II) is the major collagen of articular cartilage. Col II is synthesized as a procollagen with the amino (N)-terminal (PIINP and PIIANP) and carboxy (C)-terminal (PIICP; also referred to as CPII) domains removed during assembly of the molecule. Therefore, enzyme-linked immunosorbent assays (ELISA) to detect these in urine or serum using antibodies to PIINP/PIIANP/PIICP have been designed to indicate recent Col II synthetic activity or increased collagen synthesis as a sign of abnormal cartilage metabolism. In a 4-year study to investigate the prognostic value of PIICP, synovial fluid levels of PIICP were found to correlate well with radiographic progression of knee OA.4 However, detection of PIINP (produced primarily by mature chondrocytes5) and PIIANP (identical to PIINP except that it has an additional 69 amino acid, cysteine-rich domain and is produced by chondroprogenitor cells and dedifferentiated, pathological chondrocytes) provides an additional dimension in that changes in PIIANP:PIINP ratios could be a reflection of the pathological state of the cartilage. The NPII assay (designed to measure peptides derived from the Col II N-propeptide with the antibody detecting different peptides than the PIIANP assay) demonstrated that N-terminal procollagen type II derived peptide levels are indeed higher in human plasma and urine from patients with radio-graphically confirmed OA,6 suggesting that detecting Col II–derived peptides indicative of synthesis is a viable option for biomarkers. However, these potential biomarkers have not been validated for clinical use in OA diagnosis or monitoring treatment.
The biochemical biomarkers to date that have received a lot of attention and have shown promise in diagnosing OA effectively are primarily those that detect Col II breakdown. Of these, C-terminal cross-linked telopeptide of Col II (CTX-II), which can be detected in urine in humans by an ELISA, is the most characterized and appears to be the most promising as a noninvasive biomarker for monitoring OA.7 It provides specific and sensitive data regarding Col II breakdown and has found acceptance in OA and rheumatoid arthritis (RA) as a biomarker for joint structure changes. It has been demonstrated to correlate well with power Doppler ultra-sound synovitis and bone mineral density loss that are early markers of inflammatory arthritis.8 However, as several OA and RA patients demonstrate normal levels of uri-nary (u) CTX-II, CTX-II alone may not be diagnostically useful. Furthermore, CTX-II was detected in calcified cartilage–bone interface besides the articular cartilage matrix,9 suggesting that the tissue origins of CTX-II are not completely understood. However, levels of CTX-II have been demonstrated to correlate well with total body burden of osteophyte, a major pathological feature of OA, suggesting that CTX-II can be a component of a biomarker panel for clinical use in OA.10 In practice, CTX-II changes are often monitored in combination with other biomarkers, and its use has found success in several pharmacological trials, though in monitoring primarily hip and knee OA. As such, by combining markers of both Col II synthesis (via use of PIIANP) and collagen breakdown (via use of CTX-II), it was shown that patients with knee OA who had the largest uncoupling between Col II synthesis (low levels of PIIANP) and Col II degradation (high levels for CTX-II) had an eightfold more rapid progression of joint damage than other patients,11 suggesting that combining these two biomarkers would be effective in identifying subjects with high risk for progressive knee OA. CTX-II was also used to monitor the beneficial aspects of orally treating knee OA patients with salmon calcitonin (sCT).12 In this study it was monitored in combination with other noteworthy biomarker candidates for OA such as matrix metalloproteinase (MMP)-3 (considered a significant predictor of joint space narrowing), along with the collagenase MMP-13 (enzyme that degrades Col II), all of which demonstrated significant decreases on sCT intake. CTX-II alone has also found success in well-controlled studies to identify OA patients with high cartilage turnover and also in monitoring pharmacological intervention of OA with glucosamine sulfate.13
Besides CTX-II, other Col II breakdown products have also been used as biomarkers in monitoring OA, such as TIINE, Coll 2–1, and Coll 2–1NO2 in both serum and urine in immunoassays. Urinary TIINE is a Col II neoepitope and unlike CTX-II has the advantage that the exact nature of the immunoreactive epitope is known and has been characterized by mass spectrometry.14 It is produced by the action of several MMPs, such as MMP-13, and this study demonstrated that an MMP-13 inhibitor can reduce the levels of the TIINE neoepitope, suggesting its utility as a biomarker in monitoring drug efficacy. Assays that detect Coll 2–1 and its nitrated form, Coll 2–1NO2, provide information on oxidative related helical unwinding or further breakdown of the triple helical region of Col II and have been found to be increased in patients with primary knee OA.15,16 These relatively new biomarkers have, however, found only limited success in monitoring pharmacological trials.
Biomarkers Derived from Aggrecan
Although monitoring Col II–derived degradation products has been a primary focus of investigators, several aggrecan breakdown products informative of OA pathological conditions may have potential for application as biomarkers. Given the fact that loss of proteoglycan staining in the articular cartilage in models of OA is almost immediate, aggrecan-derived biomarkers could potentially be effective for early detection of OA. In fact, aggrecan breakdown resulting in production of the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan17 has recently been demonstrated to be detectable in human synovial fluid, serum, and urine, with the second-generation BC3-C2 antibody in a sensitive immunoassay18 opening up avenues for aggrecan-derived neoepitopes being considered as worthy bio-markers for OA diagnosis and as clinical end points for disease- modifying OA drugs. The degenerative aggrecan breakdown products AGG1 (G1–1H11) and AGG2 (6D6-G2) have been used in combination with CTX-II to validate elevated levels of synovial fluid–derived visfatin as indicative of degenerative cartilage changes during knee OA.19 Likewise, in female patients with knee OA, AGG1 and AGG2 correlated well with synovial fluid adiponectin, though no correlation was observed with CTX-II.20 Immunoassays designed to detect a complex of fibronectin (another cartilage component) and aggrecan in synovial fluids have found applications in monitoring knee pain due to meniscal injury.21 Fluctuations in the serum levels of CS846, a derivative of the chondroitin sulfate side chains of aggrecan, indicative of both aggrecan synthesis and turnover, have been shown to be associated with joint space narrowing, though it did not show promise in a pharmacological trial.22 But significant decreases in serum levels of keratan sulfate (from the keratan sulfate side chains of aggrecan) in patients with knee OA treated with chondroitin sulfate have been demonstrated, suggesting the usefulness of keratan sulfate in monitoring OA pharmacological trials and in the utility of chondroitin sulfate in treating knee OA symptoms.23
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