Metastases of advanced malignant disease to bone are common

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1 1318 Combination of Early Bisphosphonate Administration and Irradiation Leads to Improved Remineralization and Restabilization of Osteolytic Bone Metastases in an Animal Tumor Model Robert Krempien, M.D. 1 Peter E. Huber, M.D., Ph.D. 2 Wolfgang Harms, M.D. 1 Martina Treiber, M.D. 1 Michael Wannenmacher, M.D., D.D.S. 1 Burkhardt Krempien, M.D. 3 1 Department of Clinical Radiology, University of Heidelberg, Heidelberg, Germany. 2 Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center, Heidelberg, Germany. 3 Department of Pathology, University of Heidelberg, Heidelberg, Germany. Supported by a grant from the University of Heidelberg (Heidelberg, Germany). The authors thank Dr. Kalender, Dr. Engelke, and Dr. Karolczak (Institute of Medical Physics, University of Erlangen, Erlangen, Germany) for their cone-beam microtomographic analysis of the bone specimens. The authors also thank Mrs. Meixner for her excellent technical assistance. Address for reprints: Robert Krempien, M.D., Department of Clinical Radiology, University of Heidelberg, INF Heidelberg, Germany; Fax: (011) ; robert_ krempien@med.uni-heidelberg.de Accepted April 11, 2003; revision received June 3, 2003; accepted June 17, BACKGROUND. The goal of the current study was to analyze the combined effect of bisphosphonates (BPs) and irradiation on remineralization and restabilization of osteolytic bone metastases in an animal tumor model. METHODS. Bone metastases were induced in male Wistar rats via intraosseous injection of the Walker carcinosarcoma 256B cell line into both proximal tibia metaphyses on Day 1 of the study. Three treatment groups were analyzed. All animals received a single radiation dose of 17 grays (in the form of 6-megaelectronvolt electrons) on Day 7 and were sacrificed on Day 49. Group 1 (the control group) was treated with irradiation only. Groups 2 and 3 received additional BPs (clodronate; daily intraperitoneal injection dose, 20 mg/kg per day). In Group 2, BPs were given before irradiation, on Days 3 6; this schedule later was referred to as early BP treatment. In Group 3, BPs were administered simultaneously with irradiation, on Days 7 10; this schedule later was referred to as simultaneous BP treatment. The endpoints of the study were bone density and microstructural parameters of bone on Day 49. Bone density was measured using X-ray absorption. Microstructural parameters of bone were assessed using histomorphometry. A total of thirty tibiae were analyzed in each group. RESULTS. After irradiation, bone density was significantly higher among animals in the early BP treatment group compared with those in the control group and those in the simultaneous BP treatment group (P 0.001). Histomorphometric analysis of bone showed significantly better-preserved (P 0.001) microstructural parameters (bone area, trabecular number, and trabecular separation) in the early BP treatment group compared with the control and simultaneous BP treatment groups. CONCLUSIONS. Early BP administration in combination with irradiation led to improved remineralization and restabilization of osteolytic bone metastases in an animal tumor model. Cancer 2003;98: American Cancer Society. KEYWORDS: bone metastases, radiotherapy, bisphosphonates, tumor model. Metastases of advanced malignant disease to bone are common and frequently lead to morbid complications related to the skeletal system. Although the incidence of bone metastases varies between 23% and 84%, depending on the site of origin of the primary malignancy, 1 as many as 80% of patients with malignancies of the breast, prostate, or lung can be expected to develop bone metastases. 2 Morbidities associated with bone metastases include pain, pathologic fractures, and spinal cord compression. The main goals of the management of bone metastases are pain relief and skeletal stabili American Cancer Society DOI /cncr.11646

2 Irradiation and BP in Bone Metastases/Krempien et al zation with preservation or restoration of function and quality of life. Several double-blind, placebo-controlled prospective trials documenting the osteoprotective effect of bisphosphonates (BPs) in decreasing skeletal complications associated with malignant disease have been published. 3 5 Experimental studies of tumor osteopathy have demonstrated the ability of BPs not only to delay osteolytic bone lesions but also to preserve the three-dimensional microstructure of bone. 6,7 For patients with painful or unstable bone metastases, radiotherapy plays an important role, 8 bringing about pain relief and enabling the remineralization of bone lesions. 9 Osteolytic bone lesions are remineralized after local radiotherapy due to an increase in bone mass, but the weight-bearing threedimensional microarchitecture of bone is not fully restored. 10 Bone fragility is influenced by bone mass, bone microarchitecture, and tissue quality. 11 Despite the remineralization of bone lesions after radiotherapy, fractures still can occur. 12 Therefore, remineralization after successful radiotherapy may not lead to a commensurate increase in stability. BPs are used to prevent the development of bony metastases and increase bone mass in patients with malignant disease. 13 To our knowledge, before the current study, no study had undertaken to determine the efficacy of BPs combined with radiotherapy in the treatment of bone metastases. 14 The goal of the current study was to analyze the combined effect of BPs and irradiation on remineralization and restabilization of osteolytic bone metastases in an animal tumor model. The central questions to be answered were whether the osteoprotective effects of BPs, which preserve the structural integrity of bone microarchitecture, were present and whether BP administration in combination with irradiation had an additional effect on the remineralization of bone metastases. MATERIALS AND METHODS Animal Procedures One hundred six 3-month-old male Wistar rats (obtained from the Charles River, Sulzfeld, Germany), each weighing approximately 0.2 kg, were used in the current study. Thirty rats were used for ascites tumor production, 25 were used for preliminary experiments (dose finding and testing the feasibility of the system), and 5 received no injection of tumor cells and no treatment. The remaining 51 rats were used for the main portion of the study and were divided into 3 groups of 17 rats. Rats were kept in pairs at 24 C and exposed to alternating 12-hour cycles of light and darkness. All rats received a standard rat chow (Altromin, Lage, Germany) and tap water ad libitum. Food consumption and body weight were monitored at weekly intervals; no difference in these parameters was observed between groups during the course of the experiment. The guidelines provided by the German National Research Council and by German national law regarding the care and use of laboratory animals were followed. In addition, the current study was performed in accordance with guidelines issued by the University of Heidelberg (Heidelberg, Germany), and institutional and governmental approval regarding animal care and use were obtained. Experimental Procedures On Day 1, bone metastases were induced in male Wistar rats (weight, 0.2 kg) via intraosseous injection of Walker carcinosarcoma 256B cells (German Cancer Research Center, Heidelberg, Germany) into both proximal tibia metaphyses, as has been described elsewhere. 6 Radiographic imaging showed a visible osteolytic lesion on Day 7. A dose-finding study demonstrated that single doses greater than 14 grays (Gy) lead to complete tumor destruction and that, in agreement with Arnold et al., 15 doses greater than 20 Gy lead to impaired remineralization. Three groups, each consisting of 17 rats (34 tibial bones total) were studied. All rats were irradiated with a single dose of 17 Gy (6-megaelectron-volt electrons, generated by a Siemens KD2 linear accelerator; Siemens Medical Solutions, Concord, CA) on Day 7. To ensure a homogeneous dose distribution, bolus material was used. Experimental Design Osteolytic bone metastases were induced on Day 1 and irradiated on Day 7. The first signs of remineralization were seen on X-ray images on Day 21. After Day 35, all rats exhibited stable recalcification, with no evidence of residual tumor upon microscopic analysis. All animals were sacrificed on Day 49. To examine the combined effect of BPs and radiotherapy, three groups of rats with induced bone metastases were constructed. Clodronate (Boehringer- Mannheim, Mannheim, Germany), a BP, was administered daily via intraperitoneal injection at a dose of 20 mg/kg per day. The first group of rats served as a control group; rats in this group were treated with irradiation only to allow evaluation of the effect of radiotherapy alone. In the second group, BPs were administered before irradiation, on Days 3 6; this schedule later was referred to as early BP treatment. The treatment regimen for this group was designed to determine whether preservation of the weight-bearing three-dimensional microstructure in metastatic bone lesions led to quicker and more physiologic rebuilding of metastatic bone lesions after irradiation. In the third group, BPs were administered concurrently with

3 1320 CANCER September 15, 2003 / Volume 98 / Number 6 irradiation, on Days 7 10; this schedule later was referred to as simultaneous BP treatment. This regimen was designed to examine whether BPs had an additional, direct effect on the remineralization and restabilization of metastatic bone lesions after the structural integrity of the affected bone had been compromised. Two rats from each group underwent cone-beam microtomographic imaging. The remaining 45 rats (15 rats [30 tibial bones] per group) were used for bone densitometry and histomorphometry. Bone Densitometry Bone density was analyzed using quantitative radiology. Quantitative radiology involved X-ray absorption measurements of tibial metaphyses, as has been described elsewhere. 6 The X-ray absorption values for the tibiae of untreated rats (i.e., those that underwent neither tumor injection nor treatment) was taken to be the expected norm and compared with the results from the three experimental groups. Measurements were made on Day 7 (i.e., at the time of irradiation) and on Day 49. Histomorphometry Histomorphometric microarchitectural parameters are significantly correlated with reduced bone stability and pathologic fractures. 16,17 All histomorphometric parameters are presented as two-dimensional terms and were calculated and expressed according to the recommendations of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee. 18 Measurements of structural parameters in tibial cancellous bone were made with an automatic image analysis system (VIDAS; Carl Zeiss, Oberkochern, Germany) connected to a stereo microscope (Carl Zeiss) via a television camera (Bosch, Stuttgart, Germany). All measurements involving the automatic image analysis system were performed on sections stained with Masson Goldner solution. Two sections per proximal tibia were analyzed. The area 0.5 mm from the growth plate was excluded from all measurements. Specimens for measurement had an average area of approximately 15 mm 2 in each section. Imaging analysis automatically determined the measuring area (tissue area [TAr]), bone area (BAr), bone perimeter, and number of trabeculae. Based on these data, the structural parameters BAr% (equal to 100% BAr/TAr), trabecular number (TbN), and trabecular separation (TbSr) were calculated. The parallel plate model was used in the calculation of TbN and TbSr. 18 Cone-Beam Microtomography To visualize 3-dimensional bone architecture, which is known to have a sizable impact on the stability of bone, 6 rats (2 from each treatment group) were examined using 3-dimensional X-ray cone-beam microtomography (voxel size, 20 m; spatial resolution, 40 m). 19 Statistical Analysis Statistical analysis was performed with the Sigmaplot/ SigmaStat software package (SPSS, Chicago, IL). Data were analyzed using one-way analysis of variance (ANOVA). If ANOVA performed across all 3 groups indicated a significant difference (P 0.05) among the groups, the statistical differences between each pair of groups subsequently were evaluated using the Tukey test or the Dunnett multiple comparison test. A P value of less than 0.05 was considered significant. Data are presented as mean values standard deviations. RESULTS Microscopic evaluation of osteolytic bone metastases at the time of irradiation (Day 7) showed complete bone marrow infiltration by the tumor in all rats. Due to the osteoprotective effect of BPs, the trabecular bone structure was far better preserved in the early BP treatment group than in the control group (Fig. 1). At the time of irradiation (Day 7), all 3 treatment groups exhibited significantly decreased bone density (P 0.001) compared with untreated animals (i.e., those that underwent neither tumor injection nor treatment). There was no significant difference among the control, early BP treatment, and simultaneous BP treatment groups (46.5% vs. 57.4% vs. 48.4%, respectively) (Fig 2A). On Day 49, there still was a significant difference between the untreated group and the control and simultaneous BP treatment groups (100% vs. 57.6% vs. 56.7%, respectively; P 0.001). In the control and simultaneous BP treatment groups, no significant increase in bone density was observed after radiotherapy. In contrast, rats in the early BP treatment group experienced a significant increase in bone density (P 0.001) after irradiation. Consequently, there was no significant difference in bone density between the untreated group and the early BP treatment group after irradiation (100% vs. 88.1%, respectively) (Fig. 2B). Histomorphometric analysis of bone (Table 1) showed significantly better-preserved microstructural bone parameters (P 0.001) in the early BP treatment group compared with the control and simultaneous BP treatment groups. Bone area and trabecular number were significantly higher among rats in the early

4 Irradiation and BP in Bone Metastases/Krempien et al FIGURE 2. Bone density of the proximal tibia on (A) Day 7 (i.e., at the time of irradiation) and (B) Day 49. The X-ray absorption of untreated rats (i.e., those that underwent neither tumor injection nor treatment) was taken to be the expected norm (100%) and then compared with the X-ray absorption results from the 3 treatment groups. Each bar represents the mean and standard deviation of the absorption values for 30 tibial bones. *: P 0.001; No treatment: untreated group; Group 1: control group; Group 2: early bisphosphonate treatment group; Group 3: simultaneous bisphosphonate treatment group. BP treatment group compared with those in the control and simultaneous BP treatment groups. No significant difference in these parameters was found between the control and simultaneous BP treatment groups (bone area: P 0.24; trabecular number: P 0.22). Trabecular separation was significantly greater in the control and simultaneous BP treatment groups Š FIGURE 1. Bone structure of rats sacrificed on Day 7 (i.e., at the time of irradiation). All rats exhibited complete marrow infiltration by the tumor. Due to the osteoprotective effect of bisphosphonates, bone structure was much better preserved in (B) early bisphosphonate treatment group animals than in (A) control group animals.

5 1322 CANCER September 15, 2003 / Volume 98 / Number 6 TABLE 1 Histomorphometric Microarchitectural Parameters of Remineralized Osteolytic Bone Lesions on Day 49 a Variable Group 1 Group 2 Group 3 BAr% b TbN (mm 1 ) b TbSr ( m) b c Group 1: control group; Group 2: early bisphosphonate treatment group; Group 3: simultaneous bisphosphonate treatment group; BAr%: bone area as a percentage of total measurement area; TbN: trabecular number; TbSr: trabecular separation. a Each entry lists mean value standard deviation for 30 tibial bones (from 15 rats). b P for comparison with control group and comparison with simultaneous bisphosphonate treatment group. c P 0.05 for comparison with control group. than in the early BP treatment group (P 0.001). In addition, rats in the control group had significantly greater trabecular separation than did rats in the simultaneous BP treatment group (P 0.02). Microtomographic examination showed better preservation of the weight-bearing three-dimensional trabecular microarchitecture of irradiated bone metastases in rats in the early BP treatment group compared with those in the control and simultaneous BP treatment groups (Fig. 3). DISCUSSION The goal of the current study was to analyze the combined effect of BPs and irradiation on the remineralization and restabilization of osteolytic bone metastases. The results of the study demonstrated that in an animal tumor model, BP treatment aimed at preserving the structural integrity of bone facilitates a faster and more physiologic rebuilding of osteolytic bone lesions after radiotherapy. Radiotherapy of bone metastases is quite effective in alleviating pain and bringing about the remineralization of bone. Despite the remineralization of bone lesions after radiotherapy, fractures still can occur. In a Radiation Therapy Oncology Group trial involving noninterventional palliation of bone metastases, fractures were reported in 6% of all spinal sites and 13% of all long bone sites, despite the use of radiotherapy. 12 Bone fragility is influenced by bone mass, bone microarchitecture, and tissue quality. 11 Normally, bone is continually remodeled to maintain its structural integrity and stability. Tumor cells that have metastasized to bone cause a disruption in the normal interaction between osteoclasts and osteoblasts. 20 This disruption results in an uncoupling of bone remodeling, with the subsequent imbalance between bone destruction and new bone formation 21 leading to FIGURE 3. X-ray cone-beam microtomographic images of bone on Day 49 from (A) an early bisphosphonate (BP) treatment group animal and (B) a control group animal. Comparison of the two images showed that bone structure in the irradiated osteolytic lesion (O) was well preserved only in the specimen from the early treatment group, due to the osteoprotective effect of BP. After irradiation, similar bone growth and structure were observed in the newly grown trabecular bone near the growth plate. The arrow points to a dense line representing the BP-induced increase in bone density at the time of BP treatment, marking the border of the irradiated osteolytic lesion (O) at the epiphysial growth plate at the time of treatment. the loss of the weight-bearing three-dimensional microstructure of the affected bone. 22 Immediately after radiotherapy (and the resulting destruction of tumor cells), bone remodeling begins. The metastatic site is cleared of bone fragments, and new bone begins to form. Due to the loss of bone microstructure, remi-

6 Irradiation and BP in Bone Metastases/Krempien et al neralization occurs mainly within the fibrous scar tissue that fills the bone defects or along the remaining bone trabeculae, by apposition of woven bone. 23 Thus, the weight-bearing three-dimensional microstructure is not quickly and completely re-established. Because bone microarchitecture is significantly correlated with reduced bone stability and pathologic fractures, 16,17 increased bone density alone may not lead to a commensurate increase in stability after successful radiotherapy. 24 Therefore, protection of the threedimensional microarchitecture is an important therapeutic goal; achievement of this goal can help prevent bone fractures and facilitate quick restabilization. Because metastatic bone disease is mediated by osteoclastic bone resorption and by factors that stimulate osteoclastic bone resorption, it seems logical to consider the use of inhibitors of bone resorption to prevent the development of bone metastases or to delay their progression. 20 BPs are potent inhibitors of osteoclastic bone resorption. The current study, in agreement with other experimental studies, 6,7 demonstrated that BPs can preserve the structural integrity of osteolytic bone lesions even when bone density has decreased significantly. Preservation of the weight-bearing three-dimensional microarchitecture of bone significantly improves the ability of osteolytic bone lesions to remineralize and restabilize after radiation-induced tumor cell death. BPs not only inhibit osteoclastic bone resorption but also increase bone mineral density and bone mass. 11,13 Therefore, we set out to examine whether simultaneous administration of BPs had an additional effect on the remineralization and restabilization of irradiated bone metastases after the destruction of structural integrity. Simultaneous administration of BPs did not significantly enhance the therapeutic effects of radiotherapy with respect to remineralization and most microstructural parameters of bone; the only significant difference involved trabecular separation. The remodeling process after radiation-induced tumor cell death includes not only remineralization but also osteoclastic clearing of partially destroyed bone trabeculae. 25 This clearing process often initially results in additional loss of bone microstructure. 26 The significant difference in trabecular separation between the control and simultaneous BP treatment groups may result from this inhibition of osteoclastic activity during the healing process after radiotherapy. In conclusion, the current study demonstrated that in an animal tumor model, BPs in combination with irradiation led to improved remineralization and restabilization of osteolytic bone metastases. Early BP treatment geared toward preservation of the structural integrity of bone facilitates a faster and more physiologic rebuilding of osteolytic bone lesions after radiotherapy. REFERENCES 1. Rubin P. Current concepts in cancer: metastases and disseminated cancer. Int J Radiat Oncol Biol Phys. 1976;1: Coleman RE, Rubens RD. The clinical course of bone metastases from breast cancer. Br J Cancer. 1987;55: Hortobagyi GN, Theriault RL, Porter L, et al. Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. Protocol 19 Aredia Breast Cancer Study Group. N Engl J Med. 1996;335: Paterson AH, Powles TJ, Kanis JA, McCloskey E, Hanson J, Ashley S. Double-blind controlled trial of oral clodronate in patients with bone metastases from breast cancer. J Clin Oncol. 1993;11: Berenson JR, Rosen LS, Howell A, et al. Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases. Cancer. 2001;91: Krempien B, Manegold C. Prophylactic treatment of skeletal metastases, tumor-induced osteolysis, and hypercalcemia in rats with the bisphosphonate Cl 2 MBP. Cancer. 1993;72: Sasaki A, Boyce BF, Story B, et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res. 1995;55: Blitzer PH. Reanalysis of the RTOG study of the palliation of symptomatic osseous metastasis. Cancer. 1985;55: Koswig S, Budach V. [Remineralization and pain relief in bone metastases after different radiotherapy fractions (10 times 3 Gy vs. 1 time 8 Gy). A prospective study]. Strahlenther Onkol. 1999;175: Wachenfeld I, Sanner G, Bottcher HD, Kollath J. [The remineralization of the vertebral metastases of breast carcinoma after radiotherapy]. Strahlenther Onkol. 1996;172: Turner CH. Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int. 2002;13: Tong D, Gillick L, Hendrickson FR. The palliation of symptomatic osseous metastases: final results of the study by the Radiation Therapy Oncology Group. Cancer. 1982;50: Papapoulos SE. The role of bisphosphonates in the prevention and treatment of osteoporosis. Am J Med. 1993;95:48S 52S. 14. Hillner BE, Ingle JN, Berenson JR, et al. American Society Of Clinical Oncology guidelines on the role of bisphosphonates in breast cancer. American Society of Clinical Oncology Expert Panel. J Clin Oncol. 2000;18: Arnold M, Stas P, Kummermehr J, Schultz-Hector S, Trott KR. Radiation-induced impairment of bone healing in the rat femur: effects of radiation dose, sequence and interval between surgery and irradiation. Radiother Oncol. 1998;48: Legrand E, Chappard D, Pascaretti C, et al. Trabecular bone microarchitecture, bone mineral density, and vertebral fractures in male osteoporosis. J Bone Miner Res. 2000;15: Vukmirovic-Popovic S, Colterjohn N, Lhotak S, Duivenvoorden WC, Orr FW, Singh G. Morphological, histomorphometric, and microstructural alterations in human bone metastasis from breast carcinoma. Bone. 2002;31:

7 1324 CANCER September 15, 2003 / Volume 98 / Number Parfitt AM, Drezner MK, Glorieux FH, Kanis J. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1993;2: Engelke K, Karolczak M, Lutz A, Seibert U, Schaller S, Kalender WA. High spatial resolution 3D X-ray conebeam microtomograpy [abstract]. Radiology. 1999;213(P): Guise TA, Mundy GR. Cancer and bone. Endocr Rev. 1998; 19: Orr FW, Lee J, Duivenvoorden WC, Singh G. Pathophysiologic interactions in skeletal metastasis. Cancer. 2000;88: Kurth AA, Muller R. The effect of an osteolytic tumor on the three-dimensional trabecular bone morphology in an animal model. Skeletal Radiol. 2001;30: Taube T, Elomaa I, Blomqvist C, Beneton MN, Kanis JA. Histomorphometric evidence for osteoclast-mediated bone resorption in metastatic breast cancer. Bone. 1994;15: Hordon LD, Raisi M, Aaron JE, Paxton SK, Beneton M, Kanis JA. Trabecular architecture in women and men of similar bone mass with and without vertebral fracture: I. Twodimensional histology. Bone. 2000;27: Seeman E. Pathogenesis of bone fragility in women and men. Lancet. 2002;359: Mosekilde L. Consequences of the remodeling process for vertebral trabecular bone structure: a scanning electron microscopy study (uncoupling of unloaded structures). Bone Miner. 1990;10:13 35.