Diagnostic value of urinary pyridinoline for determining bone metastasis in patients with non-metastatic breast cancer Metastaz bulunmayan meme kanserli hastalarda idrar piridinolin ’ in tanısal değeri

J Clin Exp Invest www.clinexpinvest.org Vol 2, No 4, December 2011 Yazışma Adresi /Correspondence: Dr. Sevilay Sezer Ankara Numune Training and Research Hospital, Biochemistry Lab., Ankara, Turkey E-mail: sevilaysezer@gmail.com Geliş Tarihi / Received: 18.08.2011, Kabul Tarihi / Accepted: 19.10.2011 Copyright © Klinik ve Deneysel Araştırmalar Dergisi 2011, Her hakkı saklıdır / All rights reserved Klinik ve Deneysel Araştırmalar Dergisi / 2011; 2 (4): 420-424 Journal of Clinical and Experimental Investigations doi: 10.5799/ahinjs.01.2011.04.0085


INTRODUCTION
Breast cancer is the cancer of breast tissue.Worldwide, it is the most common form of cancer in females, affecting approximately 1 out of 11-12 wom-en at some stage of their life in the Western world.Although significant efforts are made to achieve early detection and effective treatment, about 20% of all women with breast cancer will die from the disease, and it is the second most common cause of cancer deaths in women. 1 Up to one-third of patients with early stage breast cancer will eventually die from the disease, and most of these (~80%) will have bone metastases. 2,3lthough a majority of these bone metastases are destructive or osteolytic, a significant percentage also causes abnormal bone formation or osteosclerotic lesions.Once tumor has metastasized to bone, the disease is incurable.Because the average survival of breast cancer patients following diagnosis of bone metastases is 24-36 months, the morbidity of bone pain, fracture, hypercalcemia and nerve compression syndromes are longstanding.Therapeutics to treat and prevent these devastating complications of bone metastases are therefore in great demand. 4e metastases to bone causes accelerated bone resorption both from direct effects of the tumor itself and through the activation of osteoclast cells in bone via humoral and growth factors such as cytokines, platelet-derived growth factor and parathyroidrelated protein. 5east cancer may affect bone metabolism, producing increased bone resorption with or without an associated increase in bone formation.Bone metastases usually stimulate bone resorption more than bone formation and thus markers of bone formation, e.g.alkaline phosphatase, are not as valuable markers of disease activity as markers of bone degradation such as urinary pyridinoline (uPYR) and urinary deoxypyridinoline (uDPD). 6YR and uDPD is formed during the posttranslational phase of collagen synthesis and is corporated into the bone matrix as a component of collagen molecules.Whereas uPYR is widely distributed throughout the body tissues, uDPD is only found in collagen of bone and dentin. 7,8en bone resorption occurs, osteoclastic degradation of bone matrix releases uPYR and uDPD into the circulation; these compounds are than excreted in urine.Measurement of urinary elimination of these compounds may provide useful information on the change of bone resorption. 9 this study, we measured uPYR and uDPD in patients without metastatic breast cancer and examined the role of uPYR and uDPD as biochemical markers of bone metastases.We compared the results of urine uPYR, uDPD and serum alkaline phosphatase (sALP) with breast cancer cases with no known bone metastases, with control subjects.And patients were followed for six years.Further-more, the effect of menopause was investigated on levels of urine uPYR, uDPD and sALP.

Patients and study design
As shown in Table I, thirty-four patients with histologically verified breast cancer without bone metastasis and 40 age-and menapausal-matched healthy control participated in the investigation.Healthy control subjects were ambulatory population without evidence of neoplastic or metabolic disease.
Patients with skeletal, endocrine, hepatic disease or with previous medical conditions or drug terapies that may affect bone metabolism were excluded from this study.Based on clinical, radiological and sintigraphic evidence, patients without metastatic breast cancer were selected for this study.All patients were referred from newly diagnosed and not receiving any treatment patient groups.
Written informed consent was obtained from both healthy controls and cancer patients before the collection of blood or urinary samples.The study was approved by the local ethics committee.

Sample collection and assays
Urine sample for measurement of uPYR, uDPD and creatinine was collected between 10:00 a.m. and 12:00 a.m., from fasting individuals.Specimens were divided into two aliquots, and urinary concentrations of creatinine were measured immediately after sample collection by standard laboratory methods using an autoanalyzer (Abbott, Aeroset -Texas, USA).The other aliquot was stored at -20°C until analyzed.
uPYR and uDPD were assayed by automated HPLC measurements was supplied by Chromosystems Instruments and Chemical GmbH, Munich, Germany.The intra-assay coefficients of variation for uPYR and uDPD were 3,8% and 5,9%, respectively, and for inter-assay variation were 5,1% and 8,9%, respectively.The values obtained were corrected for creatinine excretion and the data were expressed pmol PYR (or DPD) per µmol creatinin.
Blood samples for measurement of sALP were collected from patients who had fasted overnight, centrifuged and assayed by standard laboratory methods (Abbott, Aeroset -Texas, USA)

Statistical analysis
Results having normal distribution are reported as mean ± SD.Kolmogorov-Smirnov was used to

DISCUSSION
Bone metastasis is present in 69% of patients with terminal breast cancer.It is often difficult to detect early bone metastasis and to assess their therapeu- tic responses solely based on bone scans and bone x-rays. 9 this study, we measured urine uPYR, uDPD and sALP in patients without metastatic breast cancer and examined the role of uPYR and uDPD as a biochemical marker of bone metastasis.We compared the levels of uPYR and uDPD and sALP with breast cancer cases with no known bone metastasis, and a group of control subjects.Furthermore, the effect of menopause was investigated on levels of uPYR, uDPD and sALP and all patients were followed-up for six years.
Bone metastasis usually stimulate bone resorption more than bone formation and thus markers of bone formation, e.g.alkaline phosphatase (except prostat cancers), are not as valuable markers of disease activity as markers of bone degredation such as uPYR and uDPD.Some researchers has reported that the sensitivity of markers of bone resorption was 50-80%. 6,10Bombardieri et all.showed that sALP was not as valuable markers of disease activity as markers of bone degradation in their study. 11veral works have shown that assays of uPYR and uDPD may be valuable markers of bone resorption in a small series of patients with bone metastasis.Hiraide et al. and Walne et al. have reported elevated values of uPYR and uDPD in patients with bone metastasis compared with normal control groups. 12,13In this study, the uPYR and uDPD values for patients without bone metastasis were significantly higher than those of healthy women.
The effect of menopause on uPYR and uDPD has been assessed by several studies.Uebelhart et al. 14 showed that a significiant increase of uPYR and uDPD in postmenopausal healthy women compared to age-matched premenopausal women.Hiraide et al. 12 also showed significiantly higher values after menopause.According to these results, menopause effect should be taken into account in the diagnosis of bone metastasis by uPYR and uDPD analysis.
Although age-related changes of uPYR and uDPD values were noted in some reports, which showed significiant increase in children and postmenopausal women, Hou MF et al. 9 found that there was no significiant relationship between urine Dpd/Cre ratios and the menopausal status.This suggests that the bone destruction induced by the menopause is less than that caused by cancer metastasis.This study also showed that there were no different values of uPYR, uDPD and sALP between postmenopausal healthy women and age-matched premenopausal women.
Unexpectedly some researchers found higher levels of uPYR and uDPD in breast cancer patients without bone metastases compared to control groups' values. 8,16,17,18We have also obtained similar results.Despite a negative radiological or radioistopical skeletal survey, there are different explanations about high levels of uPYD and uDPD in patients without metastatic bone disorders.First, the invading neoplastic disease might directly induce the release of pyridinium cross-links from extraosseous tissue, such as tendons, cartilage or blood vessels.Thus, excretion in the urine of piridinyum cross-links increased and urinary PYR / DPD ratio altered.Catabolic events associated with the neoplastic disease might stimulate bone resorption throughout the skeleton.This theory is supported by the fact that mediators of tumor cachexia, such as tumor necrosis factor-α and interleukin -1 and -6 are also considered potent stimulators of osteoclast activity.Subclinical osteolysis could also contribute to the elevated levels of urinary crosslinks. 8cherstorfer et al. 8 reported that from a group of 55 individuals without bone metastasis at the beginning of the follow-up, only 2 of them had developed bone metastasis.We also followed our patients.After a 6-year follow-up of patients, 5.9% had died (n=2), 2.9% of those patients had local metastasis (n=1), 2.9% had liver metastasis (n=1), 5.9% had lung metastasis (n=2) and 8.8% had bone metastasis (n=3).
The efficacy of uPYR, uDPD to discriminate between patients without neoplastic bone involvement and healthy group was assessed by ROC analysis.Pecherstorfer et al. 8 found that the urinary pyridinium cross-links were obviously most effective in predicting the absence or evidence of metastatic bone disease.They identified that using 50 µmolPYR/ mol creatinine as the cut-off level, the sensitivity of uPYR to predict bone metastases was 88.7%, and the specificity was 41.8 %.Walls et al. showed that the sensitivity of uPYR was 80 %, the specificity was 93% by using 70 µmolPYR/ mol creatinine as the cut-off level.
We have demonstrated that the area under the ROC curve was 0.84 for uPYR, 0.79 for uDPD.So, the best diagnostic efficiency by comparing the area under the curve was provided by uPYR followed by uDPD and sALP.The cut off value, sensitivity and specificity of PYR were established 47.3 pmol/µmol creatinin, 82% and 80% respectively.The cut off value, sensitivity and specificity of uDPD were determined 9.53 pmol/µmol creatinin, 76%, 72% respectively.As mentioned above, because of the low specificity of uPYR was the release of pyridinium cross-links from extraosseous tissues after the invasion and subclinical osteolysis. 8The higher sensitivity of uPYR was considered to be due to the fact that the concentration of uDPD was about a quarter that of uPYR, so that the error in measuring uDPD assay became larger. 12 conclusion, this study revealed that measurement of uPYR and uDPD assays may contribute to the early detection of metastatic spread to bone in breast cancer patients.The high uPYR and uDPD level may be an early sign of occult metastases in patients with no bone metastasis assessed by scintigraphic techniques.
Because survival after diagnosis of bone metastases is relatively short for patients with breast tumors, methods are needed to predict skeletal complications in a shorter timescale than is possible with current imaging methods.From the results of our current study, the use of bone marker data may make a major contribution to this need by identifying those patients at highest risk who warrant the highest priority for intervention to prevent skeletal complications.But longitudinal studies should be performed to evaluate whether these parameters might be useful in monitoring progression or remission of neoplastic bone disease.

Figure 1 .
Figure 1.Receiver operating characteristic curves of uPYR, uDPD and sALP in the diagnosis of bone metastasis.

Table 1 .
Age and laboratory parameters in normal controls and cancer patients according to menopausal status

Table 2 .
Urinary pyridinoline/creatinin and deoxypyridinoline/creatinin and serum alkaline phosphatase levels in patients without bone metastases and healthy controls NS: Not significant