Received 1 March 2002; accepted for publication 28 July 2002

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1 British Journal of Haematology, 2003, 120, Comparison of five biochemical markers of bone resorption in multiple myeloma: elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for early progression of the bone disease during standard chemotherapy Niels Abildgaard, 1 Kim Brixen, 2,3 Jens E. Kristensen, 4 Erik F. Eriksen, 2 Johan L. Nielsen 1 and Lene Heickendorff 5 Departments of 1 Haematology and 2 Medicine and Endocrinology, Aarhus University Hospital, Aarhus, 3 Department of Medicine and Endocrinology, Odense University Hospital, Odense, and Departments of 4 Diagnostic Radiology and 5 Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark Received 1 March 2002; accepted for publication 28 July 2002 Summary. Increased osteoclastic bone resorption is the major causal factor of bone disease in multiple myeloma. Recently, non-invasive methods have been developed for the estimation of bone resorptive activity. To evaluate the biological sensitivity and clinical usefulness of five biochemical assays for measuring the C-terminal telopeptide of collagen I (ICTP) in serum (b-crosslaps ELISA and ICTP radioimmunoassay) and urinary creatinine-adjusted excretions of pyridinoline (PYR), deoxypyridinoline (DPD) and N-terminal telopeptide of collagen I (Ntx), we performed a study of 34 consecutive newly diagnosed myeloma patients. Serum and morning-fasting, second-void urine samples were taken before the start of treatment. In total, 40 age- and sexadjusted healthy individuals served as controls. Results were expressed as Z-scores. All test variables were highly significantly elevated in the patients (P <0Æ001). Serum (S)-ICTP was elevated (Z-score > 2) in most patients (85%) and showed significantly higher Z-score values than the other markers. S-ICTP remained more sensitive than the urinary assays when patients with impaired renal function were excluded from analysis. S-ICTP and the urinary metabolites correlated significantly with skeletal morbidity. S-b-Crosslaps correlated with the bone morbidity only when patients with renal insufficiency were excluded from the analysis. High levels of S-ICTP and urinary (U)-Ntx correlated with an increased risk for early progression of bone lesions during standard melphalan prednisolone treatment. U-Ntx and S-ICTP are sensitive tools for estimating the increased bone resorption in multiple myeloma and are clinically useful for identifying patients with increased risk of early progression of bone disease. Keywords: multiple myeloma, osteolytic bone destructions, bone resorption, carboxy-terminal telopeptide of collagen type I, amino-terminal telopeptide of collagen type I, pyridinolines. The major clinical manifestations of multiple myeloma (MM) are bone pain, pathological fractures and hypercalcaemia, due to an increased osteolytic activity. The monoclonal plasma cells produce, probably in collaboration with the stromal cells, osteoclast activating factors (for a review, see Croucher & Apperley, 1998). Even patients with no apparent clinical bone disease seem to have an increased bone resorption. However, in such patients increased bone resorption is counterbalanced by increased bone formation (Bataille et al, 1989, 1990). Correspondence: Dr Niels Abildgaard, MD, Department of Haematology, Aarhus University Hospital, Amtssygehuset, DK-8000 Aarhus C, Denmark. niels.abild@dadlnet.dk The bone disease in MM is usually assessed by X-rays of the skeleton. X-rays are useful in the diagnosis of osteolytic lesions, but do not give any dynamic information on the ongoing bone resorption. Analyses of the urinary excretions of calcium and hydroxyproline do not seem to be of sufficient value in MM (Taube et al, 1992). Information on the dynamic processes in bone may be obtained by recently introduced assays for analysis of degradation fragments from collagen type I (for a review, see Eriksen et al, 1995). Collagen type I constitutes the most dominant part of organic bone matrix and, when degraded, immunospecific antigens are liberated from the telopeptide regions at both ends of the molecule. Furthermore, the pyridinium compounds, pyridinoline (PYR) and deoxypyridinoline (DPD), originating from cross-links between Ó 2003 Blackwell Publishing Ltd 235

2 236 N. Abildgaard et al the individual collagen molecules, are liberated as free pyridinolines or bound to peptide fragments. The telopeptide fragments, PYR and DPD, circulate in the blood and are excreted in the urine. Assays for measuring both the N-terminal and the C-terminal telopeptides, and the pyridinolines have been established. We have recently shown that the serum levels and urinary excretions of these markers correlate with the osteoclastic resorptive activity in patients with MM as estimated by quantitative bone histomorphometry (Abildgaard et al, 2000). Other studies have shown that these markers are commonly elevated in MM and that they correlate with the radiologically assessed skeletal morbidity at diagnosis (Elomaa et al, 1992; Abildgaard et al, 1994; Carlson et al, 1999; Pecherstorfer et al, 1997; Fonseca et al, 2000; Woitge et al, 2001). However, no studies have compared multiple markers or established these as predictors of future bone events in patients with MM. Most studies have evaluated an radioimmunoassay (RIA) for the C-terminal telopeptide of collagen I (ICTP) (Risteli et al, 1993). This RIA measures in serum a large cross-linked fragment (> Da), which is liberated by matrix metalloproteinase activity (Sassi et al, 2000). Recently, a new serum assay measuring the C-terminal telopeptide has been introduced (b-crosslaps) (Rosenquist et al, 1998). This assay recognizes a b-isomerized aspartate-containing octapeptide within the C-terminal telopeptide. The assay has been shown to be superior to the ICTP assay in monitoring patients with metabolic bone diseases (Rosenquist et al, 1998), but has so far not been evaluated in MM. The purpose of this study was to compare the performance of five biochemical markers in identifying the increased osteolytic activity in patients with untreated MM, and to test their clinical usefulness for prediction of progressive bone involvement in MM patients. PATIENTS AND METHODS Patients. In total, 34 consecutive patients with newly diagnosed MM comprising 19 women (median 73 years, range 43 83) and 15 men (median 70 years, range 51 80) were included between November 1992 and January Most patients had advanced disease according to the Durie & Salmon staging system (stage II: six patients; stage III: 28 patients) (Durie & Salmon, 1975); 25 patients had MM of the IgG isotype, five patients had the IgA isotype, three patients had light chain disease, and one patient had nonsecretory disease. In total, 11 patients presented with hypercalcaemia (serum calcium (albumin corrected) 2Æ60 mmol/l). Four patients had elevated serum creatinine levels ( 130 lmol/l) and 12 patients had estimated creatinine clearances (creacl) < 50 ml/min {creacl ¼ [(140-age) kg)/p-creatinine]}. Two patients had normal radiological findings of the skeleton. All other patients had lytic bone lesions and/or pathological fractures. None of the patients had the rare variant with osteosclerotic lesions. None of the patients had a history of osteoporosis or other metabolic bone diseases, and none of the patients had received treatment with corticosteroids or bisphosphonates before the study. None of the patients had other malignant disease, liver disease or rheumatic disease. Serum and urine samples were taken at the time of diagnosis after rehydration but before start of treatment. Urine was collected as second-void spot samples after an overnight fast and stored at )20 C. Serum samples were taken in the morning and stored at )80 C. Both urine and serum samples were assayed at the same time at the end of the study to avoid interassay variability. All patients had symptomatic disease and received standard treatment with cyclic melphalan prednisolone (MP) given every 6 weeks until maximal response was obtained (at least eight cycles) or until registered treatment failure. Four patients received oral pamidronate 300 mg daily as part of a randomized clinical study (Brincker et al, 1998). This study showed no clinical benefit for the patients, probably due to its low intestinal absorption. These four patients were therefore not excluded from the present study. A control group consisting of 40 healthy age-adjusted individuals (20 men and 20 women) was selected. All controls had normal levels of serum creatinine. Serum and second-void morning urine samples were taken from the controls and analysed for ICTP and b-crosslaps in serum, and PYR, DPD, Ntx, and creatinine in urine. Assays. Serum concentration of C-terminal telopeptide of collagen I was analysed by two different assays: (1) the ICTP radioimmunoassay from Orion Diagnostica (Oulunsalo, Finland), which uses rabbit polyclonal antibodies detecting the pyridinoline cross-linked ICTP (Risteli et al, 1993); and (2) the automated b-crosslaps electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany) for Roche Elecsys analyser, which uses two monoclonal murine antibodies recognizing b-8aa octapeptides within the C-terminal telopeptide (Rosenquist et al, 1998). The intra- and interassay error, expressed as coefficient of variance (CV), were below 6% and 8% for ICTP, and below 4% and 6% for b-crosslaps respectively. Urinary concentrations of PYR (U-PYR) and DPD (U-DPD) were measured according to a previously published procedure (Eyre et al, 1984; Black et al, 1988) utilizing an external standard prepared from hen femora. Urine samples were hydrolysed, and the cross-links were extracted by chromatography on cellulose. The extracted cross-links were separated isocratically by reversed-phase high-performance liquid chromatography (HPLC) and identified by their fluorescence on a spectrophotometer. The intra- and interassay CVs were below 10% for both PYR and DPD. The results were expressed relative to creatinine (nmol/mmol). Urinary concentration of the Ntx (U-Ntx) was measured by an inhibition enzyme-linked immunosorbent assay (inhibition ELISA) (Hanson et al, 1992) from Ostex (Seattle, WA, USA). The assay uses a mouse monoclonal antibody (mab) 1H11, which recognizes an epitope embodied in the N-telopeptide pyridinoline region of human type I collagen. Intra- and interassay CVs were 5% and 12% respectively. Ntx results were expressed as nmol of bone collagen equivalent per mmol of creatinine.

3 Urinary hydroxyproline was measured using a commercial kit (Organon Teknica, BV Boxtel, The Netherlands). Plasma and urine creatinine, serum calcium and albumin were analysed according to standard laboratory methods. Serum calcium was corrected for individual variations in serum albumin using the formula: adjusted serum calcium (mmol/l) ¼ serum calcium total (mmol/l) + 0Æ00086 (650 ) serum albumin (lmol/l)). Bone morbidity. The severity of the bone disease at diagnosis was assessed with X-rays of the whole skeleton and scored semiquantitatively as earlier described (Abildgaard et al, 1996). The skeleton was divided into seven separate regions: cranium, cervical spine, thoracic spine, lumbar spine, pelvis, long bones and other bones. The group labelled as other bones comprised costae, sternum, claviculae, scapulae and mandible. Each region was scored according to the following staging system: no lesion was assigned score 0; one small (< 10 mm) lesion, score 1; one large (> 10 mm) lesion, score 2; multiple small lesions, score 3; and multiple mixed small/large or large lesions, score 4. The presence of radiologically assessed osteopenia was also given score 1 and added to an overall osteolysis score. Similarly, a fracture score was calculated by adding scores for vertebral collapses and non-vertebral pathological fractures. Vertebral fractures were scored according to the degree of deformity whether crashed, wedged or biconcave: less than 20% collapse was considered not pathological and assigned score 0; 20 40% collapse was given score 1; 40 60% collapse, score 2; and > 60% collapse, score 3. A nonvertebral fracture was assigned score 1. The sum of osteolysis score and fracture score made a total X-ray score. The whole skeleton was re-examined every 6 months and additionally when indicated by clinical symptoms. A progressive bone event (PBE) had by definition occurred when one of the following events were recorded: (1) development of new osteolytic lesions and/or > 25% growth of existing lesions; (2) new vertebral or non-vertebral pathological fractures or > 25% progression of existing vertebral fractures; and (3) need of irradiation and/or surgery on bone. These radiological scorings were performed blindly by one of the authors. Statistical analysis. For comparison of the test parameters, the values for ICTP, b-crosslaps, PYR, DPD, and Ntx were transformed into Z-scores, which, for each parameter Five Biochemical Bone Markers in Multiple Myeloma 237 represented, the number of standard deviations above or below the calculated mean for the control group. For each parameter of bone resorption the observed Z-scores were compared with the expected value of zero using the Wilcoxon signed rank test. The observed Z-scores for the five variables were compared by Friedman s test and differences between the individual parameters were tested by the Wilcoxon matched pairs signed rank test. Correlation between two parameters was estimated by the Spearman rank correlation analysis and the results expressed as the Spearman s correlation coefficient. The ability of the biochemical markers of bone resorption to predict progression of the bone disease was tested in univariate analysis using the Kaplan Meier product-limit estimate of the survivor function and the log-rank test for significance. For multivariate analysis, we used the Cox proportional hazards model (Cox, 1972). Values were entered both as categorical (cut-point levels at Z-score 2 versus > 2) and continuous variables, and the most significant of the two presentations was included in the model. Significant prognostic factors were entered by forward selection. A P-value less than 0Æ05 was considered significant. The statistical analyses were performed using the statistical package for social sciences (spss) version 10Æ0/PC computer program (SPSS, Chicago, IL, USA). RESULTS Biological sensitivity Data on the biochemical markers of bone resorption are presented in Table I, whereas transformed values are depicted as Z-scores in Fig 1. All variables were significantly elevated in the MM patients when compared with the control group (P < 0Æ001). Serum (S)-ICTP was elevated (Z-score > 2) in 85% of the patients, S-b-Crosslaps were elevated in 74%, U-PYR in 65%, U-DPD in 53%, and U-Ntx was elevated in 62% of the patients. S-ICTP was elevated in a significantly higher fraction (P < 0Æ05) of the patients than S-b-Crosslaps and the urinary metabolites, and S-ICTP had significantly higher Z-scores (median 5Æ9) than S-b-Crosslaps (median 3Æ4), U-PYR (median 3Æ0), U-DPD (median 2Æ8) and U-Ntx (median 2Æ9) (P < 0Æ001). In our study population, four patients had S-creatinine levels > 130 lmol/l and 12 patients had calculated Table I. Five biochemical markers of bone resorption in 34 newly diagnosed untreated patients with multiple myeloma. Variable Range 25 pc Median 75 pc Lower* Median* Upper* Serum ICTP (mg/l) 3Æ0 22Æ1 5Æ0 6Æ7 8Æ9 2Æ0 2Æ9 4Æ6 Serum b-crosslaps (mg/l) 0Æ25 3Æ2 0Æ60 0Æ83 1Æ28 0Æ12 0Æ34 0Æ59 Urine Ntx (nmol/mmol crea) Æ Urine PYR (nmol/mmol crea) Æ Urine DPD (nmol/mmol crea) Æ Æ3 10Æ7 19Æ5 43Æ8 Left: range, 25 percentile (pc), median, and 75 percentile for the patients; *right: lower reference limit, median, and upper reference limit for the age-matched control group of 40 healthy individuals.

4 238 N. Abildgaard et al Table II. Correlations between skeletal bone morbidity at diagnosis and five biochemical markers of bone resorption in 34 patients with multiple myeloma. Osteolysis score Total X-ray score All patients Serum ICTP 0Æ56** 0Æ46** Serum b-crosslaps 0Æ30 (NS) 0Æ32 (NS) Urine Ntx 0Æ53** 0Æ49** Urine PYR 0Æ56** 0Æ56** Urine DPD 0Æ49** 0Æ51** Fig 1. Z-score values for five markers of bone resorption in 34 patients with untreated multiple myeloma (MM). Boxes show median and 25 75% percentiles. Line extensions show 95% confidence intervals (CI). All markers were significantly elevated in the MM patients when compared with the control group (CI from )2 to 2). Serum C-terminal telopeptide of collagen I (ICTP) had significantly higher Z-scores than the other markers. Patients with S-creatinine < 130 lmol/l Serum ICTP 0Æ64** 0Æ63** Serum b-crosslaps 0Æ44* 0Æ50** Urine Ntx 0Æ60** 0Æ63** Urine PYR 0Æ57** 0Æ60** Urine DPD 0Æ54** 0Æ58** The Spearman correlation coefficient is shown for the whole study population (above) and for 30 patients with normal serum creatinine levels (< 130 lmol/l) (below). *Correlation is significant at the 0Æ05 level; **correlation is significant at the 0Æ01 level; NS, not significant. creacl < 50 ml/min. When patients with impaired renal function were excluded from analysis, S-ICTP continued to have significantly higher Z-score values than the urinary metabolites but this was not significantly different from S-b-Crosslaps (not shown). Correlation with radiological bone morbidity at diagnosis Serum-ICTP and the urinary variables correlated significantly with bone morbidity at diagnosis as assessed by radiography (Fig 2). No significant correlation was found between S-b-Crosslaps and the radiological scorings. Two patients with normal skeletal surveys had normal levels of all bone markers (Z-scores < 2). The correlations between the resorptive markers and the X-ray scorings are shown in Table II. The correlation coefficients increased slightly when patients with S-creatinine levels > 130 lmol/l were excluded from the analysis and, in that setting, S-b-Crosslaps correlated significantly with the X-ray scorings (Table II). Similar r-values were observed when analysis was performed for patients with creacl > 50 ml/min (not shown). Fig 2. Correlations between radiologically assessed bone morbidity at diagnosis and five markers of bone resorption in 34 patients with untreated multiple myeloma. The three columns for each marker (1 3) represent the lower, middle, and upper levels for each marker separated by cut-off points at the 33Æ3% percentiles. Boxes show median and 25 75% percentiles. Line extensions show range. P-values are indicated in the figure: *significant at the 0Æ05 level; **significant at the 0Æ01 level; NS ¼ not significant. See text for definition of X-ray scoring.

5 S-calcium (r ¼ 0Æ30, P ¼ 0Æ12) and U-hydroxyproline (r ¼ 0Æ33, P ¼ 0Æ08) did not correlate with the X-ray findings. Five Biochemical Bone Markers in Multiple Myeloma 239 Prediction of progressive bone events In total, 32 patients were observed for up to 30 months (mean 18 months). One patient was lost to follow-up and one patient died unexpectedly at home soon after the first admission; 25 patients had at least one progressive bone event during the observation period. The criteria for progressive bone disease were met as follows: need for radiotherapy (one patient); new vertebral collapses (nine patients); progression of lytic lesions (eight patients); and simultaneous occurring new vertebral collapses and progression of lytic lesions (seven patients). Elevated levels of U-Ntx were significantly correlated with an increased risk for early progression of the bone disease (Table III). S-ICTP, and marginally, elevated S-Crosslaps, also seemed to correlate with an increased risk of early progression of the bone disease (Table III), but normal levels were only observed in few patients. Excluding patients with S-creatinine levels > 130 lmol/l did not affect the prognostic significance of elevated levels of U-Ntx (P ¼ 0Æ03), ICTP (P ¼ 0Æ02), or Crosslaps (P ¼ 0Æ05). Nine patients had primary progressive disease during MP treatment. These patients did not have higher pretreatment levels of the biochemical markers than patients who responded to MP treatment. Patients with elevated U-Ntx at diagnosis had an approximately 50% risk of progression of the bone disease within the first year of MP treatment, whereas new bone events were only observed in approximately 20% of the patients with normal pretreatment U-Ntx (Fig 3). In univariate analysis, other baseline parameters predictive for early progression of the bone disease were high total X-ray score, high fracture score, high osteolysis score, Durie and Salmon stage III disease, and elevated S-b2-microglobulin levels. Fig 3. Kaplan Meier estimate of the bone event-free survival in MM patients with normal pretreatment urinary levels of Ntx ( 119 nmol/mmol crea) (13 pts, dotted line) versus in patients with elevated pretreatment U-Ntx (> 119 nmol/mmol crea) (19 pts, solid line). Censored pts are marked by. All patients were treated with standard oral melphalan prednisolone for at least 1 year or until disease progression. The significant factors in univariate analysis were included into the multivariate Cox analysis. U-Ntx as a continuous variable showed the highest significance and was the first factor to enter the Cox model. S-ICTP as a continuous variable had a similar chi-square value (6Æ9 versus 7Æ0 for Ntx). Owing to a correlation between U-Ntx and S-ICTP, the chi-square value for ICTP was reduced after the entering of U-Ntx by forward selection. However, S-ICTP was included into the model as the only other factor of independent predictive value (Table IV). The model estimates an approximately 35% relative risk increase for an early progressive bone event if pretreatment U-Ntx was increased by 100 units, e.g. from 100 to 200 nmol/mmol crea, and similarly an approximately 15% relative risk increase for an Table III. Bone resorptive markers as predictors of early progression of bone disease in patients with multiple myeloma (MM) treated with melphalan prednisolone. Variable Cut-off level No. patients Median time to first bone event (months) P-value U-Ntx (nmol/mmol crea) ± 3 0Æ02 > Æ5 ± 3 S-ICTP (lg/l) 4Æ ± 4Æ5 0Æ02 >4Æ Æ5 ±4Æ5 S-b-Crosslaps (lg/l) 0Æ ± 4Æ5 0Æ06 >0Æ Æ5 ±4Æ5 U-PYR (nmol/mmol crea) ± 3 0Æ28 > ± 2 U-DPD (nmol/mmol crea) 43Æ ± 4Æ5 0Æ40 >43Æ ± 3 Cut-off levels were defined at mean + 2SD for a control group of 40 healthy, age-matched individuals. P-value according to the log-rank test.

6 240 N. Abildgaard et al Table IV. Cox proportional hazards model for the value of base-line parameters for prediction of progressive bone events within the first year after diagnosis in 32 MM patients treated with standard chemotherapy. Variable b SE Relative risk P-value U-Ntx (nmol/mmol crea) 0Æ0035 0Æ0016 1Æ0035 0Æ01 S-ICTP (lg/l) 0Æ14 0Æ0627 1Æ15 0Æ02 SE, standard error. Relative risk for a variable is the relative risk for the increment of one unit of the observed parameter. early progressive bone event if pretreatment S-ICTP was increased by 1 unit, for example from 5 to 6 lg/l. DISCUSSION Increased bone resorption is observed in almost all patients with untreated MM. Even patients with no apparent radiological bone disease have increased bone resorption (Bataille et al, 1990). The ideal biochemical marker of bone resorption should therefore fulfil the following criteria: (1) be elevated in most patients with newly diagnosed MM; (2) reflect the severity of the actual resorptive activity; and (3) provide relevant information for making decisions on therapy in clinical practice. In our study, all markers were significantly elevated in patients with MM. S-ICTP was elevated in a higher fraction of the patients and showed higher levels than the other markers. ICTP in serum is cleared by renal excretion (Risteli et al, 1993), and elevated serum levels of ICTP could therefore partly be explained by decreased renal function, which is often seen in patients with MM. However, excluding patients with estimated creatinine clearance below 50 ml/min, we found that S-ICTP remained significantly elevated and showed the highest Z-score values. We found that all biochemical markers except S-b-Crosslaps correlated with a quantitative radiological scoring of the skeletal morbidity. The biochemical markers and radiographs, however, do not give the same information. The markers reflect the speed of ongoing resorptive activity in bone, whereas the X-rays give a time-point status, summarizing the amount of bone resorption that has occurred to date. Thus, a close relationship between X-rays and biochemical markers is not to be expected. In our untreated symptomatic patients, however, the highest levels of the biochemical markers were observed in those patients with the most abundant bone disease. We have recently shown that U-Ntx, U-DPD and S-ICTP correlate with the bone resorption rate as assessed by dynamic bone histomorphometry (Abildgaard et al, 2000). Together, these data indicate that U-Ntx, U-DPD and S-ICTP do reflect the severity of the ongoing bone degradative activity in MM. The ICTP assay has been found to be quite insensitive in reflecting bone resorption and effects of antiresorptive treatment in metabolic bone diseases (Garnero et al, 1994; Rosenquist et al, 1998). A number of observations, however, indicate that S-ICTP may be of more value in MM. It has recently been established that the antigenic determinant of the ICTP assay resides within phenylalaninerich regions of the C-terminal telopeptides of the two a1 chains (Sassi et al, 2000). An intact trivalent cross-linked structure of the telopeptide is necessary for optimal immunoreaction. Immunogenicity is lost after protease degradation with cathepsin K, but preserved after treatment with matrix metalloproteinases (MMP). Both cathepsin K and MMP-9 are proteases involved in osteoclastic bone resorption, but the contribution of each protease may be modified by different pathophysiological conditions. It has been suggested that an intracellular pathway involving phagocytosis and lysosomal (cathepsin K) degradation of collagen mainly functions during balanced turnover states, whereas an extracellular pathway involving MMPs is particularly active under conditions of rapid remodelling, for example, in cancer-induced states (van der Everts et al, 1996). Interestingly, a study has shown that myeloma cells can express MMP-9 and induce upregulation of MMP-1 and MMP-2 in the stromal environment in co-culture systems (Barille et al, 1997). The b-crosslaps assay detects a b-isomerized aspartate containing octapeptide within the C-terminal telopeptide region of collagen I (Rosenquist et al, 1998). b-isomerization is believed to be associated with the ageing of proteins, and has been demonstrated to occur over time within the peptide sequence of the C-terminal telopeptide region of collagen I (Fledelius et al, 1997). This octapeptide sequence is not degraded by cathepsin K (Sassi et al, 2000). Thus, the b-crosslaps assay may be particularly useful for monitoring the resorption of relatively old bone as part of the remodelling process, and this assay has also been shown to be superior to the ICTP assay in monitoring metabolic bone diseases (Rosenquist et al, 1998). However, as discussed above, the b-crosslaps assay might not be the best suitable assay for monitoring bone resorption in MM. In this study, elevated pretreatment levels of U-Ntx and S-ICTP, and marginally S-b-Crosslaps, were predictive for early progression of the bone disease. U-Ntx and S-ICTP were the only factors to enter the Cox model. All patients received the same standard treatment of cyclic MP for at least one year, and patients who responded by traditional criteria to the MP treatment had similar pretreatment levels of the markers as patients with primary progressive disease.

7 So, elevated levels of U-Ntx and S-ICTP are not just pretreatment indicators of MP resistance, but seem to be independent, prognostic factors for the development of the bone disease. However, it should be noted that the number of patients in our study was rather small, and the results need to be confirmed in a larger group of patients. In newly diagnosed MM patients, U-Ntx and S-ICTP seem to reflect an overall high bone resorptive activity and bone degradative capacity that cannot be suppressed sufficiently by standard MP treatment. Whether these patients could benefit from high-dose chemotherapy or treatment with intravenous bisphosphonates cannot be determined by our data, but need further studies. From our data, U-Ntx and S-ICTP seem to be good candidates as useful markers of bone resorption in MM. The potential usefulness of such markers is obvious. In MM, bone degradative activity may proceed even during good remission phases of the disease (Abildgaard et al, 1997). The introduction of bisphosphonates has been shown to be important in the treatment of the bone disease in MM (Berenson et al, 1998). However, the best regimen for using bisphosphonates in MM has still to be determined. Furthermore, the treatment should be tailored for each patient. In this context, a valid non-invasive marker of increased bone resorption is mandatory. Our study indicates that U-Ntx and S-ICTP are useful and sensitive markers of bone disease in patients with MM. ACKNOWLEDGMENTS Kirsten Hald and Mette Carstens are thanked for their skilful technical assistance. The study was supported by grants from the Institute of Experimental Clinical Research, Aarhus University, and from the Frits, Georg, and Marie Gluds Foundation. Five Biochemical Bone Markers in Multiple Myeloma 241 REFERENCES Abildgaard, N., Nielsen, J.L. & Heickendorff, L. 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