Bone markers as diagnostic tools
Bone scintigraphy is considered to be the gold standard for monitoring metastatic bone involvement.22 However, since scintigraphy is expensive, lacks specificity and is not particularly suitable for the follow-up of patients, bone markers as indicators for bone metastasis in PCa patients have been studied.2, 3, 4, 5, 6 There are no clear recommendations which markers or marker combinations should be used.9 Analyses were performed in both serum and urine, but consistent results were not always described. The preanalytic variability of bone markers, their different stability in vitro especially in urine and use of assays with different antibodies recognizing different epitopes may explain many of the discrepant results.23, 24, 25, 26, 27 To avoid these discrepancies as far as possible, we measured bone markers exclusively in serum collected during a defined time period, between 7:00 and 9:00 A.M. In elderly patients, blood sampling is always more reliable and more practicable than urine sampling. As commercial assays for measuring bone formation and resorption in serum are available, measurements in urine could be replaced by those in serum/plasma.28
Our present study, to our knowledge, involves the largest number of bone turnover markers in serum of PCa patients for both diagnostic and prognostic evaluation to date. Of the 10 bone turnover markers measured, 3 of the 4 bone formation (tALP, bALP and P1NP) and resorption (BSP, TRAP and NTX) markers as well as OPG were significantly increased in PCa patients with bone metastases. These results correspond to histomorphologic evidence that not only osteoblastic but also resorptive processes occur.29 Differences between the markers could reflect the occurrence of a marker in either an early or a late period of bone formation or resorption.2 Hormonal therapy could similarly give rise to differences. Nonmetastatic PCa patients receiving antiandrogen therapy had moderately increased serum bALP,6, 30 and the type of therapy (orchiectomy, luteinizing hormone–releasing hormone analogs and/or antiandrogens) is likely to influence bone turnover.31 In our study, bone formation markers were not different between patients with and without hormonal therapy in the N1M0 or M1 groups. Only slightly increased values of the resorption markers CTX and TRAP were observed in lymph node-positive patients who received hormonal therapy. These changes did not affect the accuracy of the markers for diagnosing bone metastasis. However, since not only “fresh” but also treated patients were investigated, this limitation of our study should be considered in characterizing the diagnostic accuracy of the bone turnover markers.
Bone formation markers.
Bone ALP is a well-established bone marker. It can be considered together with tALP as a “standard” analyte when the utility of other bone markers is to be assessed and compared.2 In our study, the advantage of bALP compared to tALP became evident in a higher diagnostic sensitivity when the assay-specific upper cut-off of tALP in our hospital was used as the decision point (Table II). In contrast to bALP as an enzyme directly secreted by osteoblasts, P1NP is a metabolic indicator of bone formation. P1NP measured with a radioimmunoassay (Orion, Espoo, Finland) has been recommended as a promising bone marker in PCa patients.5, 32, 33 Our data confirm these results using a new assay that measures both tri- and monomeric P1NP forms, while the Orion radioimmunoassay detects only the trimeric form. The diagnostic sensitivity of OC was low compared to the other bone formation markers. Its appearance in the late bone formation phase in contrast to ALP or P1NP as typical markers of the early phases of osteoblastic proliferation and matrix maturation could explain the divergent diagnostic efficacy.2 In addition to the clinical validity, both assays are uncomplicated and reliable with good analytic performance; thus, they fulfill the essential preconditions for routine measurements. The P1NP assay is an automated assay on a general-purpose analyzer (Elecsys). Meanwhile, bALP can also be measured on an analyzer (Access, Beckman-Coulter), which gives results almost identical to those of the Tandem Ostase test used in our study. All of these arguments support the view that bALP and P1NP are the most suitable single bone formation markers to detect bone metastases or to confirm their absence in PCa patients.
Bone resorption markers.
Dissociation was also observed between bone resorption markers. CTX showed lower diagnostic sensitivity than NTX, TRAP and BSP (Table II). NTX had the lowest analytic performance, and the test kits were not always available. The TRAP assay used in our study is based on a combined approach of immunoassay and activity measurement. In contrast to other TRAP immunoassays, this new ELISA with reliable analytic performance detects the active isoform 5b without any interference from inactive enzyme forms or fragments.34, 35, 36 BSP is generally considered to be a bone resorption marker.37 It is overexpressed in PCa tissue and bone metastases.38, 39 As yet, only few data exist on the behavior of serum BSP in PCa patients. Sensitivities and specificities of about 90% for the diagnosis of bone metastases have been described.40 Our data were similar, though BSP was determined without disrupting its interaction with complement factor H. However, BSP was the only marker with increased concentrations already in the pN1M0 group, and numerous patients of the pN0M0 group showed increased values. As there was no further evidence for the occurrence of bone metastases in those patients, we assumed that overexpression of BSP in PCa tissue already raises the BSP concentration in serum before bone metastasis develops. Therefore, BSP should not be considered a specific bone metastasis marker in PCa as suggested for breast cancer.16 In addition, the instability of reagents and the disadvantage of a radioimmunoassay hindered determination of BSP as a routine parameter up to now. Taking these data of bone resorption markers together, we recommend TRAP as a single bone resorption marker for PCa patients.
As osteoclastogenesis is the connecting link between bone formation and bone resorption, determination of OPG and RANKL was of special interest. Both OPG and RANKL were overexpressed in bone metastases of PCa patients.10, 11 OPG has been postulated to be a survival factor in PCa cells.41, 42 We and another group found increased serum OPG concentrations with advanced PCa,8, 13 while elevations were not observed in breast, colorectal, pancreatic and renal tumor patients with bone metastases.43 The present study confirms the discriminatory power of OPG in distinguishing between PCa patients with and without bone metastases. However, despite this clinical significance, it has to be considered that OPG is also produced by a variety of other organs.44 Thus, changed serum concentrations of OPG caused by other diseases, such as vascular diseases or rheumatoid arthritis, need to be taken into account when interpreting serum OPG values.44, 45 Based on the role of OPG and RANKL,11, 41, 42 it could be assumed that the ratio of RANKL to OPG would facilitate the diagnosis of bone metastasis. However, neither serum RANKL concentrations nor the ratio of RANKL to OPG was related to metastatic stage (Fig. 1). Thus, measurement of RANKL does not appear to be helpful in this respect. Similar findings have been reported in patients with Paget's disease.46
As the univariate evaluation of data showed dissociated changes of serum marker concentrations for both bone formation and resorption as well as for those linked with osteoclastogenesis, multivariate analysis was appropriate. Logistic regression analysis of all variables showed that the combination of OPG and TRAP is the best way to differentiate between bone metastasis and nonmetastasis. The overall correct classification of 93% achieved by the combination of TRAP and OPG corresponds to the results described when the ratio of OC to P1NP or OC to bALP as a marker characteristic of different bone formation phases is used.33 As OPG alone resulted in a sensitivity and specificity >90%, a rather small improvement of diagnostic accuracy could be expected by TRAP.
Bone markers as prognostic tools
The extent of bone metastasis monitored by bone scans is a strong predictor of survival in PCa patients.47, 48 Some studies have also demonstrated this association with ALP, P1NP and CTX.12, 49, 50 PSA was not directly associated with bone progression in all studies.12, 49, 50 Univariate analyses confirmed the strong predictive value of bone markers concerning survival in our study (Table III, Fig. 3). Not only did OPG yield the highest relative risk value in the univariate analysis but OPG and BSP were the only independent predictors of PCa-specific death in the multivariate Cox model (Table III). Thus, OPG really appears more as a marker of tumor burden or activity than a simple indicator of bone turnover. The value of serum BSP as an independent prognostic factor for subsequent bone metastasis was previously demonstrated in patients with primary breast cancer.51
The current study provides data on the association between serum OPG, bone metastases and survival in PCa patients. Our results are consistent with the functional role of OPG in facilitating the survival of PCa cells by protecting them from apoptosis induced by TRAIL.41, 52 The design of our study, with the limitation that patients with hormonal pretreatment were included, did not allow an exact answer as to whether the increase of serum OPG occurs in the early phase, during or in the progression phase of metastasis.
In conclusion, our results suggest that, despite the limitations, measurement of serum OPG concentration is a powerful test alone or in combination with other bone turnover markers to detect bone metastatic spread and to predict survival probability in PCa patients. In addition, the association of OPG with the survival of PCa patients could be used for stratifying patients with advanced PCa for clinical trials in the current treatment options with bisphosphonates or other chemotherapeutic agents to individualize treatment.53, 54, 55 However, increased serum OPG caused by other organ alterations must be considered.