Biochemical Assessment of Paget's Disease of Bone

Abstract

Biochemical measurements of bone turnover provide an objective assessment of disease activity and the response to treatment. Alkaline phosphatase is the best characterized of the bone turnover markers and reflects the extent and activity of Paget's disease. However, in addition to bone-specific alkaline phosphatase (Bone ALP), there is also osteocalcin (OC) and procollagen type 1 N-terminal propeptide (P1NP) as formation markers. A variety of telopeptides (C-terminal telopeptide of type I collagen, [CTX], N-telopeptide of type I collagen [NTX]) or cross-link breakdown products of type 1 collagen can be used to assess bone resorption. Total alkaline phosphatase (Total ALP), Bone ALP, and P1NP all perform similarly in diagnosis and in evaluating the response to treatment, but the general availability, low interassay variation, and inexpensiveness of Total ALP makes it the best test for routine use. Measurement of the biological variability of the different markers in stable, untreated Paget's disease indicates how great a change (critical difference) is needed to define a true alteration in disease activity. Bone ALP, P1NP, and NTX show the highest therapy induced change/critical difference ratio during antiresorptive treatment. Some of the resorption markers show more complex changes in response to treatment. Pyridinoline (PYD) or deoxypyridinoline (DPD) cross-links of type 1 collagen are excreted in urine either as free or as peptide bound moieties, but it is the latter which decrease by the greatest amount in response to bisphosphonate therapy. Newly formed type 1 collagen contains an aspartyl-glycine motif (αCTX), which undergoes spontaneous isoaspartyl formation to βCTX as the bone ages. In untreated Paget's disease, the αCTX is raised proportionately more (16-fold) than βCTX (3-fold) and decreases in response to bisphosphonate therapy to a greater extent than βCTX (measured in the sCTX assay). As bisphosphonates have become more potent, the aim of treatment has shifted toward the achievement of a rate of bone turnover in the lower part of the reference range. This is important because the duration of remission of disease activity is strongly determined by the post treatment nadir bone turnover.

INTRODUCTION

The main clinical consequences of Paget's disease are bone pain, deformity, and fracture, but their response to treatment is often attended by considerable individual variation. In contrast, biochemical measurements of bone turnover provide an objective assessment of both disease activity and the response to treatment. Such measurements can be used to plan management to achieve optimal control of bone turnover and estimate the duration of remission with important implications for the need for retreatment. In the last few years, the availability of extremely potent bisphosphonates has made it necessary to review previous assumptions about current goals of treatment. In addition, an expansion in the range of available markers has made it easier to evaluate such responses, whereas the introduction of serum-based measurements and assays designed for the auto-analyzer has increased applicability, reduced cost, and improved precision.

BIOCHEMICAL MARKERS OF BONE TURNOVER IN PAGET'S DISEASE

Alkaline phosphatase is the best characterized of the bone turnover markers and reflects both the extent and activity of the Paget's disease but tends to be disproportionately raised with skull involvement.^{1, 2} Although the increase in Total alkaline phosphatase (Total ALP) in Paget's disease is secondary to the change in bone resorption, it has a well established role in the evaluation of disease activity as well as in the monitoring of the response to antiresorptive therapy. In addition to Bone ALP, there is also osteocalcin (OC) and procollagen type-1 N-terminal propeptide (P1NP) as formation markers with hydroxyproline (Hyp) and a variety of telopeptides (C-terminal telopeptide of type I collagen [CTX], N-telopeptide of type I collagen [NTX]) or cross-link breakdown products of type 1 collagen that can be used to assess bone resorption. It is clear that the different biochemical markers reflect a range of cellular processes, as well as providing technical challenges to the biochemist. This can be shown by the roles of OC and the telopeptide cross-links: the former has proved disappointing, whereas the latter offers considerable promise.

OC is less consistently raised in active disease compared with other formation markers such as ALP and P1NP and also shows a smaller decline with treatment.^3-5 There may be a number of reasons for this lack of sensitivity. OC is secreted by osteoblasts and, because of its high affinity for calcium and hydroxyapatite, is incorporated into the bone matrix. Some of it enters the circulation, where the half-life is short because of rapid catabolism, mainly in the kidney. It is also cleaved in the circulation by proteases such as plasmin and some cathepsins, which generate a number of fragments of varying size. OC is also released into the circulation by bone resorption, where cleavage at both C− and N-terminals results in the appearance of midmolecular fragments, although the midportion of the molecule seems resistant to cathepsin-mediated proteolysis. Rapid proteolysis in vitro and the presence of multiple fragments in serum may account for some of the measurement variability. Recent assays that measure either the intact 1–49 peptide or larger 1–43 fragments have shown improved ability to reflect bone turnover.⁶ Pagetic bone stains intensely for OC, indicating incorporation into bone in a pattern similar to normal subjects. It is unknown whether the rate of incorporation is altered in Paget's disease.⁷Altered OC synthesis by pagetic osteoblasts or increased incorporation caused by the high mineral content of woven bone with decreased release into the circulation has been suggested as explanations for the relative insensitivity of this marker to reflect the increased turnover of Paget's disease.⁸ The OC gene is regulated by 1,25-dihydroxyvitamin D, which increases in the early phase of antiresorptive therapy as bone turnover is temporarily uncoupled.^{9, 10} This may serve to maintain levels of osteocalcin and contribute to the lack of sensitivity in monitoring response.

The increase in bone resorption that is the driving force behind the biochemical and clinical manifestations of Paget's disease can be assessed from the measurement of breakdown products of type 1 collagen such as the C− or N-terminal telopeptides or the cross-links between the collagen molecules. Hydroxyproline was the traditional resorption marker but includes a significant nonbone component, is a time-consuming manual assay, and has largely been replaced by automated methods of more specific collagen degradation products.

Pyridinoline (PYD) or deoxypyridinoline (DPD) cross-links of type 1 collagen are excreted in urine either as free or peptide-bound moieties. It is the peptide-bound cross-links that decrease by the greatest amount in response to bisphosphonate therapy. The fractional clearance of free DPD (fDPD) is greater than unity, suggesting intrarenal production from degradation of peptide-bound cross-links that therefore maintains the free concentration in the presence of falling bone resorption.^{4, 11}

The C-terminal telopeptide assay is a good marker in high turnover bone disease and measures degradation products of type 1 collagen that are components of the peptide-bound cross-link fraction. There is also evidence that CTX can be used to assess changes in bone matrix quality and bone resorption. Newly formed type 1 collagen contains an aspartyl-glycine motif (αCTX), which undergoes spontaneous isoaspartyl formation to βCTX as the bone ages.¹² In untreated Paget's disease, but not in thyrotoxicosis and hyperparathyroidism, the αCTX is raised proportionately more (16-fold) than βCTX (3-fold) and decreases in response to bisphosphonate therapy to a greater extent than βCTX (measured in the sCTX assay).^{12, 13} Immunolocalization studies on bone sections from normal subjects, and a patient with Paget's disease showed that normal lamellar bone stains intensely for βCTX.¹² Woven bone in the pagetic lesion and newly deposited lamellar bone on the surface of normal osteons stained more intensely for αCTX. This observation was confirmed and extended by the demonstration of a high α/βCTX ratio in collagen digests of pagetic bone. The reduction in the relative proportions of α and βCTX as bone turnover falls with bisphosphonate treatment is consistent with a return toward a more normal maturation of matrix. The problem with CTX is that serum levels are reduced by food and thus may underestimate disease activity if measured nonfasting.¹⁴

Collagen type 1 telopeptides are also subject to isoaspartyl formation, but the specificity of the antibody used in the NTX assay for these two epitopes is uncertain.¹⁵ Other markers that have been studied include carboxy-terminal cross-linked telopeptide of type 1 collagen (1CTP) and carboxy-terminal propeptide of type 1 collagen (P1CP) but these have shown poor discriminatory value in diagnosis and monitoring even in patients with very active disease.^{4, 10, 16, 17}

Bone formation and resorption remain coupled in Paget's disease despite the increase in turnover. Although osteocalcin and βCTX are highly correlated with each other as are P1NP and bone ALP with NTX, there is a poor correlation between OC or βCTX and the other markers.⁵

Discriminatory value of biochemical markers

A number of factors influence the choice of marker including the availability of serum or urine samples and cost. A major determinant is the ability to identify abnormality and monitor the response to treatment in different clinical situations. In the pretreatment assessment of patients, the requirement is for a test that is consistently elevated across a wide range of disease activity. Total ALP, Bone ALP, P1NP, and NTX perform well in this situation, whereas βCTX, OC, and fDPD perform poorly, and the reasons for this have already been discussed (Table 1).^{5, 18}

Table Table 1.. Analytical and Biological Variation of Markers of Bone Turnover

• * Least significant change calculated from Ref. 5.
• CVa, interassay CV obtained from control values in the pathological range; CVi, coefficient of biological variation (within- and between-subject variation) from patients with stable Paget's disease.

The choice of marker to monitor the response to treatment is predominantly determined by analytical and biological variation. This can be derived from the interassay CV (CV_a) and the within-subject variation (CV_i) and incorporated into the calculation of the critical difference or least significant change , which indicates whether two sequential measurements reflect a true biological difference.^{16, 19} Bone turnover markers measured on automated platforms perform better than the manual methods, whereas serum markers show lower within-subject biological variability than urinary markers. A recent study showed the importance of establishing biological variation in active disease rather than extrapolating from normal subjects. In Paget's disease with a high bone turnover, a change in total ALP of >35% is needed to exceed the critical difference, whereas a change of only 15% is required in a normal subject.¹⁸

The other important determinant of the choice of marker is its performance in monitoring the response to treatment. In clinical practice, the requirement is for a marker that shows a substantial decrease with treatment, and this can be evaluated by expressing the magnitude of the change during treatment as a ratio to the least significant change (Fig. 1). The greater the ratio above unity, the more discriminatory the measurement. In polyostotic Paget's disease, Total ALP, Bone ALP, and P1NP all perform similarly in diagnosis and assessment of response to treatment.²⁰ The general availability, low interassay variation, and inexpensiveness of total ALP makes it the best test for routine use.^{5, 17} Several studies have shown that Bone ALP, P1NP, and NTX show the highest therapy induced change/critical difference ratio during antiresorptive treatment.^{4, 5, 20} Although urine NTX is more commonly raised in the untreated state compared with the other resorption markers such as urinary DPD or serum βCTX, it shows more biological and analytical variability in response to therapy, particularly compared with the automated serum markers.⁵

Figure Fig. 1.

Evaluation of sensitivity of bone turnover markers for monitoring the response to treatment.

Monostotic Paget's disease

The ratio of the decrease during treatment to the least significant change can also be used to compare the use of a particular marker in the evaluation of monostotic disease, where the limited extent of skeletal involvement represents a significant challenge to biochemical evaluation. Bone ALP seems to be the most sensitive marker and was increased in 60% of patients with limited disease activity/extent even though Total ALP was normal.¹⁹ In a recent study comparing markers in patients with both monostotic and polyostotic involvement, Bone ALP showed a much better response to (tiludronate) treatment than Total ALP, although P1NP was also useful for monitoring with a ratio of >2. Only NTX of the resorption markers showed a ratio >1 in the early phase of treatment, whereas Hyp, sCTX, and uCTX all had ratios less than unity.²⁰ In this study, bone turnover tended to increase in the 6 months after the completion of tiludronate therapy in patients with monostotic Paget's disease but remained suppressed in polyostotic disease. This was attributed to the weak skeletal binding of tiludronate and the limited scope for uptake and reattachment of the bisphosphonate to pagetic bone in monostotic disease.

In a recent study, αCTX proved to be very sensitive in the assessment of untreated Paget's disease and performed as well as NTX, and both were better than TRACPs, Total ALP, and Hyp.¹³ In monostotic disease, αCTX also changed to a greater extent with bisphosphonate therapy compared with other markers and may have advantages in this respect comparable with Bone ALP.²⁰

USE OF BIOCHEMICAL MARKERS OF BONE TURNOVER IN CLINICAL PRACTICE

Diagnosis and evaluation of Paget's disease

Many patients will present with focal symptoms such as bone pain, fracture, or deformity, which indicate the need for diagnostic radiography and evaluation of bone turnover. Others will present with an incidental finding of a raised ALP, where history, examination, liver function tests, and imaging such as ultrasound or CT will be needed to separate hepatic and skeletal causes of the abnormality. An isotope bone scan is the most cost-effective diagnostic test, perhaps preceded by measurement of bone resorption markers to support a skeletal origin of the raised ALP. Where liver disease has been excluded and the patient has no skeletal symptoms, there is still a need for a clear diagnosis and evaluation of the need for treatment largely determined by the height of the ALP and patient/physician anxiety.

Assessment of baseline bone turnover/disease activity

Separation of pain caused by Paget's disease from that caused by associated osteoarthritis is best done by history and examination supplemented by radiography of the appropriate bone and adjacent joint. Measurement of bone turnover may be helpful in that high values suggest the potential to improve symptoms with bisphosphonate treatment despite the presence of osteoarthritis, whereas normal values point to symptoms predominantly arising from the joint. Further discrimination may be aided by isotope bone scanning to distinguish between joint and bone uptake at the site of pain. Quantitation of the isotope uptake in a small focus of disease can be used to monitor the response to bisphosphonate treatment and compliment the smaller changes in biochemical markers (Fig. 2).

Figure Fig. 2.

Evaluation of monostotic Paget's disease with regional uptake on isotope bone scan.

Response to treatment

Bone turnover markers provide the most objective approach to the evaluation of the response to treatment, but it is critical to express these changes in a logical and robust manner. As bisphosphonates become more potent, the influence of baseline bone turnover and extent of Paget's disease, which are strong predictors of the response to treatment, become weaker.²¹ There are currently two considerations that are relevant to this issue. The first is to identify the earliest point in time at which a response can be detected, and this will influence the choice of marker. The use of a potent intravenous nitrogen containing bisphosphonate will reduce sensitive resorption markers such as NTX by days 9–10, and this can be used to predict the nadir Total ALP achieved during the subsequent year.¹⁰ Whereas this may be important in comparing different bisphosphonates in clinical trials, this rapidity of evaluation is rarely needed in clinical practice. The only exception may be where there are severe neurological compression symptoms and when early retreatment for a predicted incomplete response may avoid the need for surgical intervention. In routine clinical practice, it will generally be sufficient to measure Total ALP ∼6 weeks after the completion of treatment. This will indicate the near-final response and can be combined with the residual symptomatology to help decide whether retreatment might be needed.

The usual indication for biochemical evaluation is to determine whether the aim of treatment has been achieved. The introduction of highly potent bisphosphonates has shifted the goals of treatment away from the percentage decrease in activity to the proportion of patients who achieve normal turnover. Even here, there has been a move away from accepting values within the upper part of the reference range toward values for bone turnover at or below the midpoint of the reference range. The justification for more demanding criteria centers around what we define as normal bone turnover. The reference range for any biological measurement describes the scatter of values in a “normal” population free of (bone) disease. However, normal bone turnover for a patient with Paget's disease should mean a level of activity similar to that which existed before the development of the disease. This is usually unknown, but the lower the post-treatment bone turnover, the greater the probability that this will be similar to the pre-Paget's disease value. The other driving force for more stringent criteria for response has been the clear demonstration that the duration of biochemical remission is strongly determined by the nadir value achieved by treatment.^22-24 Although there is no objective evidence, there seems a reasonable supposition that control of long-term complications of the disease such as fracture, deformity, and degenerative joint disease might be prevented or reduced by long-term control of the disease.

Defining remission and relapse

A consequence of this approach is the need to define remission and relapse. The former might be regarded as the maintenance of normal bone turnover, but the critical test is by how much can the value change and still represent stability. With each measurement of bone turnover, there is a critical difference or least significant change as discussed earlier. Measurements within these limits represent stability, whereas values outside the least significant change represents a statistically significant increase or decrease. The values for the least significant change for the common bone turnover markers are shown in Table 1. The choice of measurement will depend on availability and cost and also on the ability of a particular bone turnover marker to perform well within the clinical context such as the advantages of Bone ALP over Total ALP in monostotic Paget's disease.

The use of clinical features with which to define relapse is attended by all the problems of their use for determining response. There are several ways of defining relapse biochemically that range in precision and practical use. The simplistic approach is to define relapse in arbitrary terms as a post-treatment value outside the reference range or as a given multiple of the upper limit of normal. This is easy to understand but is insensitive and will underestimate the true incidence of relapse. At the other end of the spectrum, relapse can be defined as an increase in a chosen bone turnover marker that exceeds the least significant change. Because most of these patients will have a bone turnover in the lower part of the normal range, the least significant change should be calculated from a normal population. This is intellectually more satisfying and is statistically sound but will identify a high rate of relapse. The clinical significance of such a small change is uncertain, as is the value of intervening with another course of bisphosphonate therapy at that particular point in time. However, as the duration of this type of biochemical remission becomes more common with the use of highly potent bisphosphonates, there may be less reluctance to retreat small increments in turnover at 3–4 yearly intervals given the hope that sustained control will prevent the emergence of long-term complications.²⁵ The choice of marker most applicable to this situation has not been systematically explored, but the experience of monitoring the response of monostotic disease would seem to indicate a preference for Bone ALP, P1NP, and NTX. An alternative, pragmatic approach would be to monitor total ALP at ∼6-month intervals in primary care and retreat a significant (15%) increase from the nadir.¹⁸ These different approaches are susceptible to testing in some of the current follow-up studies in progress.

True resistance to bisphosphonates is rare. The most common explanation is poor compliance with oral therapy, and this can be confirmed and corrected by the administration of an equipotent intravenous bisphosphonate. Very active disease may fail to respond to a single course of treatment but will be controlled by further drug.^{21, 25} True biochemical resistance does occur but is rare, and the underlying mechanism is unclear.²⁶ It is often overdiagnosed because of failure to recognize that, for individual patients, there is a minimum “normal” level of bone turnover that cannot be improved on by even the most potent bisphosphonate.

SUMMARY

Biochemical bone turnover markers provide the most objective measure of the activity of Paget's disease and the response to treatment. The range of available analytes has increased enormously over the last decade, increasing choice, reducing cost, and improving clinical use. Whereas response to treatment should be based on the least significant change or critical difference derived from a population with active Paget's disease, remission should be based on values from normal subjects. Appropriate choice of marker optimizes the ability to assess disease activity and make long-term predictions about future requirements for treatment.