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Cancer Epidemiology Biomarkers & Prevention Vol. 15, 1392-1395, July 2006
© 2006 American Association for Cancer Research

Short Communication

Alpha CTX as a Biomarker of Skeletal Invasion of Breast Cancer: Immunolocalization and the Load Dependency of Urinary Excretion

Diana J. Leeming1, Günter Delling2, Mitsuru Koizumi3, Kim Henriksen1, Morten A. Karsdal1, Bo Li1, Per Qvist1, László B. Tankó4 and Inger Byrjalsen1

1 Nordic Bioscience Diagnostics A/S, Herlev, Denmark; 2 Institute of Bone Pathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 3 Cancer Institute Hospital, Tokyo, Japan; and 4 Center for Clinical and Basic Research, Ballerup, Denmark

Requests for reprints: Diana J. Leeming, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. Phone: 45-4452-5216; Fax: 45-4454-8888. E-mail: djl{at}nordicbioscience.com


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently showed that increased urinary excretion of the cross-linked, nonisomerized form of the C-telopeptide of collagen type I ({alpha}{alpha}CTX) could be a sensitive indicator of the presence of bone metastases in breast cancer patients. The present study was sought to investigate (a) the localization of {alpha}CTX epitopes in the proximity of a bone metastasis and (b) the relationship between number of metastases and the urinary excretion of {alpha}{alpha}CTX. Adjacent bone sections from breast cancer patients were stained for the presence of tumor cells (anti-cytokeratin antibody), osteoclasts (TRAcP activity), and {alpha}CTX (anti-{alpha}CTX antibody). The association between the extent of metastatic bone disease and urinary excretion of {alpha}{alpha}CTX measured with ELISA was assessed in 90 breast cancer patients (45 with bone metastasis and 45 without bone metastasis). Immunohistochemistry revealed accumulation of TRAcP-positive osteoclasts and intense staining for {alpha}CTX epitopes in the proximity of cytokeratin-positive bone metastasis. Areas of {alpha}CTX staining showed unstructured bone tissue under polarized light. In addition, there was a significant linear association between the number of bone metastases and the urinary levels of {alpha}{alpha}CTX in breast cancer patients with metastatic bone disease, independent of age and body mass index (r = 0.56, P < 0.001). The estimated relative increases in {alpha}{alpha}CTX associated with the presence of one, two, or three metastases are 38%, 57%, and 81%, respectively. Taken into account the 17% intraindividual variation of the assay, {alpha}{alpha}CTX could be a sensitive biochemical marker for the close monitoring of cancer patients aiming the facilitation of early metastasis detection. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1392–5)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Early diagnosis of skeletal metastases in breast cancer patients has important consequences for the prognosis of the primary disease. The diagnosis of bone metastases routinely relies on skeletal X-rays and bone scintigraphy. However, limited sensitivity and potential harm of serial exposure to X-ray makes these techniques inadequate for the close monitoring of cancer patients.

Metastatic bone disease is characterized by acceleration of bone remodeling at sites of the metastases. Collagen type I constitutes the main component of the extracellular matrix. The epitope 1207EKAHGDDR1214 is located in the C-telopeptide {alpha}1 chain of collagen type I and found as an {alpha}-form in the GD motif of newly synthesized collagen (1). It undergoes spontaneous nonenzymatic ß-isomerization with aging. In a previous study, Houzé et al. showed a significant correlation between scintigraphic scores of metastatic bone invasion and urinary ßCTX levels in breast cancer patients (2). However, in pathologic situations of high bone remodeling, quantification of degradation products from the newly synthesized collagen ({alpha}{alpha}CTX) can be expected to provide an even more relevant biomarker. Indeed, three recent studies (1, 3, 4) undertaken in breast and prostate cancer patients illustrate the higher sensitivity of {alpha}{alpha}CTX for indicating the presence of bone metastases compared with ßßCTX.

The aim of this study was to investigate (a) whether {alpha}CTX epitopes are detectable in histologic sections of tumor infiltrated bone in breast cancer patients, (b) how number of bone metastases influence the urinary excretion of {alpha}{alpha}CTX in breast cancer patients compared with those without bone metastases, (c) the potentials of this marker for the early detection of skeletal invasion (one to three metastases).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
The histologic analyses were done on seven iliac crest or transpedicular bone metastasis bone biopsies of the spine of metastatic breast cancer patients from archive material in Hamburg (G.D.). Specimens were fixed in buffered formalin (4%), decalcified by ultrasonic sound in combination with constant temperature at 24°C, and embedded in paraffin. This method is in use as routine process for immunohistochemical characterization of primary or secondary bone tumors.

The use of measuring urinary {alpha}{alpha}CTX for the detection of bone metastases was assessed in 90 breast cancer patients (45 without bone metastases and 45 with bone metastases). All patients were routinely scanned by radiography at the Cancer Institute Hospital in Tokyo, Japan (M.K.). Positives for bone metastases were verified by Tc99 bone scintigraphy together with computer tomography and/or magnetic resonance imaging to verify the presence and determine the number of bone metastases as described previously by Soloway et al. (5). All patients with skeletal complications were newly diagnosed, and none of them had received therapies known to influence bone turnover for the past 2 years before entry to the study. All participants signed an approved written consent, and the studies were done in accordance with the Helsinki Declaration II and Standards of Good Clinical Practice. Local ethical committees have approved study protocols.

Histology and Immunohistochemical Localization of {alpha}CTX in Bone
Human bone specimens were decalcified and embedded in paraffin for preparation of sections with 5 µm in thickness. The sections were blocked in TBS containing 0.5% casein and incubated overnight at 4°C in a moist atmosphere with primary antibody diluted in TBS containing 0.5% casein or control without antibody. The primary antibodies employed were monoclonal; F44 raised against the {alpha}CTX epitope (Nordic Bioscience, Herlev, Denmark) and another raised against cytokeratin against pan-cytokeratin (CK-MNF). Osteoclasts were visualized by their specific TRAcP activity. After incubation, the sections were thoroughly washed in TBS and incubated with secondary peroxidase-labeled antibody (Mouse EnVision; DakoCytomation, Glostrup, Denmark) for 30 minutes at room temperature. Finally, the peroxidase activity was visualized using 3,3'-Diaminobenzidine Plus (Sigma, St. Louis, MO), and the nuclei were counterstained using Ehrlich's hematoxylin. The bone slices were then dehydrated through a gradient of alcohol (70-99%) and toluene and mounted in DPX. Pictures were taken using an Olympus BX-60 light microscope equipped with an Olympus C-5050 Zoom digital camera.

Quantification of {alpha}CTX in Urine
Second morning void urine samples was collected from all patients and stored at –40°C until analysis. The concentration of {alpha}{alpha}CTX fragments was measured by the ALPHA CTX ELISA (Nordic Bioscience, Herlev, Denmark) following the manufacturer's recommendations. This assay is a sandwich-format EIA that uses monoclonal antibodies raised against {alpha}-EKAHDDGR ({alpha}CTX) and measures cross-linked chains ({alpha}{alpha}CTX). The monoclonal antibody is highly specific for the {alpha}-form of CTX with <1% cross-reactivity with the ß-form of CTX1. Urinary excretion of {alpha}{alpha}CTX was corrected for creatinine excretion, which was measured by standard routine method. The short-term intraindividual variation of the ALPHA CTX ELISA, determined in 17 healthy postmenopausal women providing five consecutive samples during a 14-day observation period, was 17%.

Statistical Analysis
Demographics of subjects with or without bone metastases was compared with Student's t test. The concentrations of {alpha}{alpha}CTX were calculated relative to the creatinine concentration and transformed logarithmically to obtain normality. The General Linear Models Procedure was used for the regression analysis assessing the relationship between increases in {alpha}{alpha}CTX level (relative to patients without bone metastases) and the number of metastases. Model check included test for normality and probability and residual plots. The Statistical Analysis System (SAS, Cary, NC) was used for all calculations.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histology and Immunohistochemical Localization of {alpha}CTX in Bone
{alpha}CTX staining was seen throughout all seven bone sections in the presence of tumor cells and consequently a large number of active osteoclasts. Figure 1A to D shows adjacent sections from a bone invaded by breast cancer. Areas with hyperchromatic nuclei and positive cytokeratin staining confirmed the presence of malignant tissue in these sections (Fig. 1A). In the proximity of the tumor, TRAcP-positive staining revealed numerous osteoclasts characteristic for osteolytic lesions (Fig. 1B). In addition, immunostaining also revealed diffuse presence of {alpha}CTX epitopes with more intensive staining at the sites of high bone remodeling (Fig. 1C). Control using no antibody showed no staining (Fig. 1D).


Figure 1
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  		Figure 1. Histology and immunohistochemistry done on bone tumor secondary from primary breast cancer: (A) Pan-cytokeratin (CK-MNF, DAKO), (B) TRAcP-positive staining of osteoclasts, (C) immunolocalization of {alpha}CTX epitopes, (D) control (adjacent sections), (E and F) area of woven and lamellar bone in the proximity of bone metastasis and {alpha}CTX staining, (G and H), area of healthy bone structure distant from the metastatic invasion with corresponding {alpha}CTX staining. Magnification, x10 (A-D and G-H) and x60 (E-F). E and G, bone microstructure (woven versus lamellar bone) under polarized light.

 
Figure 1E shows a magnified area of bone tissue with both woven and lamellar bone structure as differentiated by polarized light. Figure 1F indicates that {alpha}CTX staining is confined to areas of woven but not lamellar bone. This latter is also illustrated by sections of fully normal bone structure: large areas of lamellar bone do not reveal presence of {alpha}CTX epitopes (Fig. 1G and H).

Urine {alpha}{alpha}CTX in Breast Cancer Patients
The demographic data of breast cancer patients with or without bone metastases has been published earlier (3). In brief, there were no statistically significant differences in terms of age or body mass index, but the urinary excretion of {alpha}{alpha}CTX was highly increased in patients with bone metastases (P < 0.001; Table 1 ).


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  		Table 1. Demographic and {alpha}{alpha}CTX data in breast cancer patients stratified by ± bone metastases

 
An aim of the study was to explore the utility of measuring {alpha}{alpha}CTX for the detection of metastatic invasion in breast cancer patients. Therefore, increases in urinary {alpha}{alpha}CTX in patients with bone metastases were expressed with reference to the mean value of patients without bone metastases. Figure 2 indicates the linear association between number of metastases and the relative increases in the urinary excretion of {alpha}{alpha}CTX in patients with distinctive metastases (n = 40; r = 0.56, P < 0.001); Superscan patients (75% of ribs, vertebrae an pelvic bone infected by bone metastases) were excluded. According to the model, the relative increases corresponding to the presence of one, two, and three metastases are estimated to be 38%, 57%, and 81%, respectively. We have previously reported that level and variance of urinary {alpha}{alpha}CTX in breast cancer patients without bone metastases and age-matched healthy controls are comparable (6).


Figure 2
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  		Figure 2. Percentage increase in {alpha}{alpha}CTX in 40 breast cancer patients +BM relative to 45 breast cancer patients –BM.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main result of the study were (a) the demonstration of the presence of {alpha}CTX epitopes in bone tissue being in the close proximity of metastatic invasion and related osteolytic activity and (b) a significant association between urinary levels of {alpha}{alpha}CTX and the extent of skeletal invasion (i.e., number of metastases) in breast cancer patients. The findings provide insights into why collagenous biomarkers, and in particular, {alpha}{alpha}CTX is a valid marker for the detection of osteolytic metastases in breast cancer patients.

Breast carcinoma frequently metastasizes to specific organs, including lymph nodes, lung, and bone. The vast majority of bone metastases in breast cancer patients is osteolytic lesions (7), although mixed and osteoblastic metastases have also been observed (8, 9). The mechanisms of intercellular communication between bone and cancer cells have been recently proposed by Clines and Guise (10). Cytokines/hormones (interleukin-1, interleukin-6, and tumor necrosis factor-{alpha}) and parathyroid hormone-related protein secreted from breast cancer cells are known to stimulate receptor activator of nuclear factor-{kappa}B ligand and reduce osteoprotegrin expression in osteoblasts. This in turn leads to enhanced differentiation of osteoclast progenitor cells and formation/activation of osteoclasts. The result of these events is the disruption of normal bone remodeling with predominance of osteoclastic bone resorption-promoting osteolysis (11).

Secondary to the increased osteoclastic bone resorption, the activity of osteoblasts will also increase and facilitate continuous formation of new bone matrix at the margin of the metastasis. At the same time, growth factors (insulin-like growth factors, transforming growth factor-ß, platelet-derived growth factor, and bone morphogenetic protein) and cytokines from osteoclasts enhance the growth and survival of tumor cells and further stimulate the expression of parathyroid hormone-related protein. These mechanisms will collectively result in the increase of a vicious cycle (12) driving the intensive remodeling. Presence of {alpha}CTX epitopes in bone tissue being in the proximity of a metastasis is a reflection of accelerated turnover of newly synthesized collagen type I. Another evidence of intensive remodeling is the lack of matured, lamellar structure in bone tissue revealing {alpha}CTX-staining. Importantly, in matured, lamellar bone tissue distant from the bone metastases, no {alpha}CTX staining can be detected. Thus, these findings indicate that {alpha}CTX is closely related to skeletal sites of intensive bone remodeling, such as bone being in the proximity of bone metastases.

Numerous efforts have been made to use biomarkers of bone formation, resorption, and osteoclastogenesis for the detection and quantification of bone metastasis from breast cancer. In our recent study (3), we provided further evidence that the collagenous bone resorption markers {alpha}{alpha}CTX, ßßCTX, NTX, and ICTP measured in serum or urine showed significant increases in breast cancer patients compared with those without bone metastasis. The largest relative increases were revealed by {alpha}{alpha}CTX with increasing differences with the advancement of the metastatic disease. Our present findings indicated a significant association between the number of metastases and the increases in urinary {alpha}{alpha}CTX relative to age- and body mass index–matched breast cancer patients without bone metastases. The estimated relative increases in {alpha}{alpha}CTX corresponding to the presence of one, two, or three metastases were 2- to 5-fold higher than the intraindividual variation. Although the present study is of cross-sectional design, the aforementioned results also nurture the notion that the indicative value of an increase in {alpha}{alpha}CTX levels for skeletal invasion can be improved when monitoring the same cancer patient with serial measurements.

In summary, our present analysis provide initial explanation to why {alpha}{alpha}CTX is a promising marker for the first-line noninvasive diagnostic of skeletal invasion in patients with known breast cancer disease.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Conflict of interest: D.J. Leeming, B. Li, P. Qvist, and I. Byrjalsen are full-time employees of Nordic Bioscience, a company engaged in the development and marketing of bone and cartilage markers.

Received 11/30/05; revised 2/27/06; accepted 3/13/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cloos PAC, Lyubimova N, Solberg H, et al. An immunoassay for measuring fragments of newly synthesized collagen type I produced during metastatic invasion of bone. Clin Lab 2004;50:279–89.[Medline]
  2. Houzé P, Bellik B, Extra JM, Bouro F, Bousquet B. Urinary carboxyterminal telopeptide of collagen I as a potential marker of bone metastases chemotherapy monitoring in breast cancer. Clin Chim Acta 1999;281:77–88.[Medline]
  3. Leeming DJ, Koizumi M, Byrjalsen I, Li B, Qvist P, Tankó LB. The relative utility of eight collagenous and non-collagenous markers for diagnosis of skeletal metastases in breast, prostate, or lung cancer patients. Cancer Epidemiol Biomarkers Prev 2006;15:32–8.[Abstract/Free Full Text]
  4. Cloos PAC, Fledelius C, Christgau S, et al. Investigation of bone disease using isomerized and racemized fragments of type I collagen. Calcif Tissue Int 2003;72:8–17.[Medline]
  5. Soloway MS, Hardeman SW, Hickey D, et al. Stratification of patients with metastatic prostate cancer based on extent of disease on initial bone scan. Cancer 1988;16:195–202.
  6. Leeming DJ, Koizumi M, Li B, et al. Non-isomerized C-telopeptides of type I collagen ({alpha}CTX): a sensitive indicator of metastatic bone disease. J Bone Miner Res 2005;20:S65.
  7. Guise TA, Mundy GR. Cancer and bone. Endocr Rev 1998;19:18–54.[Abstract/Free Full Text]
  8. Fogelman I, Cook G, Israel O, Van der Wall H. Positron emission tomography and bone metastases. Semin Nucl Med 2005;35:135–42.[CrossRef][Medline]
  9. Brown B, Laorr A, Greenspan A, Stadalnik R. Negative bone scintigraphy with diffuse osteoblastic breast carcinoma metastases. Clin Nucl Med 1993;19:194–6.
  10. Clines GA, Guise TA. Hypercalcaemia of malignancy and basic research on mechanisms responsible for osteolytic and osteoblastic metastasis to bone. Endocr Relat Cancer 2005;12:549–83.[Abstract/Free Full Text]
  11. Thomas RJ, Guise TA, Yin JJ, et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 1999;140:4451–8.[Abstract/Free Full Text]
  12. Wittrant Y, Théoleyre S, Chipoy C, et al. RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis. Biochim Biophys Acta 2004;1704:49–57.[Medline]



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