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Keywords:

  • ZOLEDRONIC ACID;
  • BONE GROWTH;
  • OSTEOSARCOMA;
  • EWING SARCOMA;
  • BONE RESORPTION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Osteosarcoma and Ewing sarcoma represent the two most frequent primary bone tumors that arise in the pediatric population. Despite recent improvement in their therapeutic management, no improvement in survival rate has been achieved since early 1980 s. Among new therapeutic approaches, bisphosphonates are promising candidates as potent inhibitors of bone resorption. However, their effects on bone growth must be studied at dosing regimen corresponding to pediatric protocols. To this aim, several protocols using zoledronic acid (ZOL) were developed in growing mice (50 µg/kg every 2 days × 10). Parameters of bone remodeling and bone growth were investigated by radiography, micro–computed tomography, histology, and biologic analyses. Extramedullar hematopoiesis was searched for in spleen tissue. A transient inhibitory effect of ZOL was observed on bone length, with a bone-growth arrest during treatment owing to an impressive increase in bone formation at the growth plate level (8- to 10-fold increase in BV/TV). This sclerotic band then shifted into the diaphysis as soon as endochondral bone formation started again after the end of ZOL treatment, revealing that osteoclasts and osteoblasts are still active at the growth plate. In conclusion, endochondral bone growth is transiently disturbed by high doses of ZOL corresponding to the pediatric treatment of primary bone tumors. These preclinical observations were confirmed by a case report in a pediatric patient treated in the French OS2006 protocol over 10 months who showed a growth arrest during the ZOL treatment period with normal gain in size after the end of treatment. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, representing more than 500 patients diagnosed every year in Europe. Survival has not evolved or improved for the past 10 to 20 years, whatever the protocol applied throughout Europe, still leading to survival rates of around 50% to 70% for patients with localized disease at 5 years.1 Current standard therapeutic approaches include preoperative chemotherapy with the use of a triple schedule of a cocktail of several drugs, including ifosfamide, VP-16, and methotrexate. Then stratification of patients with good tumor response (GR, presence of less than 10% residual malignant cells) and poor tumor response (PR, more than 10% residual tumor cells) is performed on resected tumor tissue. This Huvos grading is the main recognized prognostic factor used for the past two decades worldwide, along with the presence of metastatic disease at diagnosis, the histologic subtype, tumor volume, or tumor location. Postoperative chemotherapy then is adapted to this Huvos grading.

Ewing sarcoma represents the second malignant primary bone tumor in children. It is characterized histologically by small round cells with poorly delineated borders, and the hallmark feature of Ewing sarcoma is the presence of an aberrant transcription factor with oncogenic properties as a result of the transcription of the EWS-FLI1 fusion gene.2 The prognosis of patients with bone or medullar metastases and of patients who relapse or are resistant to initial chemotherapy is less than 25%.

Both osteosarcoma and Ewing sarcoma are characterized by rapid and extensive osteolysis. The relevance of using bone-resorption-targeting drugs as adjuvant therapies has emerged in recent years based on preclinical studies performed with bisphosphonates in animal experimental models.3–5 This concept has been translated to clinical trials in several countries both in Europe (OS2006 in France, EUROEWING in Germany) and in the United States.6

Bisphosphonates (BPs) are synthetic analogues of the naturally occurring pyrophosphate, sharing a common P[BOND]C[BOND]P structure in which the central carbon atom allows the addition of two side chains. Based on their chemical structure, BPs are broadly divided into two main families: nitrogen- and non-nitrogen-containing BPs.7 Among the nitrogen-containing BPs (N-BPs), zoledronic acid (ZOL) belongs to the third-generation BPs, characterized by a heterocyclic substituent. BPs have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function.8 The N-BPs inhibit protein prenylation via the mevalonate pathway,9 leading to decreased osteoclast activity and function.10, 11 The increase in BMD following therapy results from reduction in the remodeling space owing to decreased activation frequency of bone modeling cycles with a cumulative positive bone balance.12, 13 BPs are stable in bone for prolonged periods of time with long half-lifes in the skeleton for more than 300 days, as reported by in vivo studies in animals.14 They are used extensively in clinical practice for the treatment of disease with high bone catabolism, mainly osteoporosis, Paget disease, some skeletal pediatric disorders (eg, osteogenesis imperfecta, juvenile rheumatoid arthritis, and juvenile idiopathic osteoporosis), and cancer-related bone diseases such as multiple myeloma and breast carcinoma.15, 16

The clinical interest in using BPs in osteosarcoma has been strengthened by several preclinical studies.3, 5, 17 The potential antitumor effect observed may be explained by a direct activity on tumor cells,18 by a strong modulation of the tumor microenvironment,19, 20 or by stimulation of immune effectors.21, 22 ZOL appears to have similar inhibitory activities on Ewing sarcoma cell lines and on preclinical models developed in immunodeficient mice.4, 23, 24 Interestingly, a strong benefit that associates with BPs and conventional chemotherapy has been demonstrated by several authors for both tumors in vitro25–27 and in vivo.3, 4, 28

Overall, these preclinical data prompted the clinicians to propose ZOL as adjuvant therapy for pediatric and adult patients bearing osteosarcoma, such as, for example, in the randomized French protocol OS2006. Previous studies have reported on safety concerns about BPs use in children, but at lower doses given for pediatric osteoporosis or osteogenesis imperfecta (OI), for example.29, 30 Only one study recently reported the development of metaphyseal sclerotic bands in osteosarcoma patients treated with pamidronate as well as epiphyseal and vertebral endplate sclerosis progressing to a bone-within-bone appearance, these findings occurring more frequently in younger patients with open epiphyses.31 Very few preclinical experiments were performed and here again based on lower BP doses than those used in oncopediatric protocols.32–34

The purpose of this study was to determine the effect of high doses of ZOL, adapted from those given for pediatric osteosarcoma/Ewing sarcoma patients following a comparative time schedule in growing mice from different strains. The effects of ZOL were investigated on the parameters of bone growth by X-ray analysis, X-ray micro–computed tomography (µCT), histomorphometry, and histology.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Animals and drug administration

Animals aged under 21 days were provided by internal breeding of C57BL/6J mice initially purchased at Janvier's Breeding (Le Genest Saint Isle, France) or purchased directly at Janvier's Breeding when older than 21 days. Mice were housed under pathogen-free conditions at the Experimental Therapy Unit (Faculty of Medicine, Nantes, France) in accordance with the institutional guidelines of the French Ethical Committee and under the supervision of authorized investigators. Puberty was estimated around day 35 in the mice. Three series of animals were studied independently (Fig. 1):

  • Protocol 1: C57BL/6J mice were randomized into two groups for eight subcutaneous injections of PBS (controls) or ZOL (0.1 mg/kg in PBS, provided as the disodium hydrate by Novartis Pharma AG, Basel, Switzerland) twice a week beginning on day 7. Animals were euthanized on day 28 (end of treatment) and then 14 days and 1 and 2 months after the end of the treatment (Fig. 1, protocol 1). This dosing regimen was used previously in preclinical studies demonstrating the therapeutic relevance of ZOL in murine osteosarcoma models.31 However, these doses are more comparable with the adult dosing regimen than with the regimen for children. Indeed, adult clinical ZOL doses (4 mg intravenously every 3 to 4 weeks) are equivalent to approximately 100 µg/kg of the research-grade disodium salt used in this study.

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Figure 1. Summary of zoledronate protocols. Protocol 1: C57BL/6J mice were randomized into two groups for eight subcutaneous injections of PBS (controls) or ZOL (0.1 mg/kg in PBS) twice a week beginning on day 7. Animals were euthanized on day 28 (end of treatment) and then 14 days and 1 and 2 months after the end of treatment. This dosing regimen was used previously in preclinical studies demonstrating the therapeutic relevance of ZOL in murine osteosarcoma models. Protocol 2: C57BL/6J female mice were randomized into two groups for 10 subcutaneous injections of PBS (controls) or 0.05 mg/kg of ZOL every 2 days from days 14 to 35 (corresponding to mouse puberty). Animals were euthanized on day 35 (end of treatment) and then 14 days and 1, 2, and 4 months after the end of treatment. This dosing regimen is more comparable with pediatric protocols (10 injections of 0.05 mg/kg ZOL). Protocol 3: Two groups of female mice were randomized to receive 10 subcutaneous injections of PBS (controls) or 0.05 mg/kg of ZOL every 2 days from days 28 to 46. Animals were euthanized on day 36 (puberty), day 48 (end of treatment), and then 1, 3, and 4 months after the end of treatment. This protocol is more comparable with an osteosarcoma dose and time schedule for patients with a median age of 18 years.

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  • Protocol 2: C57BL/6J female mice were randomized into two groups for 10 subcutaneous injections with PBS (controls) or 0.05 mg/kg of ZOL every 2 days from day 14 to day 35. Animals were euthanized on day 35 (end of treatment) and then 14 days and 1, 2, and 4 months after the end of treatment (Fig. 1, protocol 2). This dosing regimen is more comparable with pediatric protocols (10 injections of 0.05 mg/kg of ZOL). We therefore adapted the time and dosing regimen to mice with a treatment endpoint at 35 days of age, corresponding to mouse puberty.

  • Protocol 3: Three mouse strains were compared (C57BL/6J, C3H/HeN, and Swiss); two groups of female mice were randomized to receive 10 subcutaneous injections with PBS (controls) or 0.05 mg/kg of ZOL every 2 days from day 28 to day 46. Animals were euthanized on day 36 (puberty), day 48 (end of treatment), and then 1, 3, and 4 months after the end of the treatment (Fig. 1, protocol 3). This protocol is more comparable with osteosarcoma dose and time schedule with a median age of 18 years for patients.

All animal groups were statistically equivalent (n = 6) with respect to body weight at the earliest time points of each study.

Tibial length and body mass

Longitudinal growth data were collected at the end of treatment and afterwards by length measurements of the left tibias made with the radiography apparatus Faxitron (Edimex, Angers, France). Data were available from each study from days 39 to 63 for protocol 1, days 37 to 61 for protocol 2, and days 28 to 74 for protocol 3 in control and ZOL-treated animals (n = 3).

The animals were weighed twice a week during each treatment and once a week after the end of treatment until the latest endpoint.

X-ray µCT analysis

Analyses of bone microarchitecture were performed using a Skyscan 1076 in vivo µCT scanner (Skyscan, Kontich, Belgium). Tests were performed both on live animals throughout the treatment and after killing on tibias taken from treated mice. For analyses performed on living animals, mice were anesthetized by an intraperitoneal injection of 10 µL/g of a Rompun (Bayer, Puteaux, France)–Imalgène 1000 (Merial, Lyon, France) solution (8% and 13%, respectively, in PBS). Six left tibias were analyzed postmortem for each treatment group.

All tibias were scanned using the same parameters (pixel size 9 µm, 50 kV, 0.5-mm Al filter, 30 minutes of scanning). The reconstruction was analyzed using NRecon and CTan software (Skyscan). The volumes of interest (VOI) were selected as 15% and 50% of the trabecular bone. Bone volume/tissue volume parameters were measured for each VOI. 3D visualizations of tibias were done using ANT software (Skyscan) at each time of interest during animal growth and at killing.

Histology

Tibias and spleens were collected from euthanized mice from control and ZOL 50 or 100 µg/kg groups (corresponding to protocols 1 to 3) and were fixed in 4% buffered formaldehyde. Tibias were decalcified in 4.13% EDTA/0.2% paraformaldehyde (pH 7.4) over 4 weeks. The specimens were deshydrated and embedded in paraffin. Then 5-µm-thick sagittal sections stained with Masson's trichrome (three-color staining protocol produce red muscle fibers, green collagen and bone, light red cytoplasm, and dark brown nuclei) were observed using a DMRXA microscope (Leica, Nussloch, Germany). Tartrate-resistant acid phosphatase (TRACP) staining was performed on tibial sections to identify osteoclasts by 1 hour of incubation in a 1 mg/mL of naphthol AS-TR phosphate, 60 mmol/L N,N-dimethylformamide, 100 mmol/L sodium tartrate, and 1 mg/mL Fast red TR salt solution (all from Sigma Chemical Co., St Louis, MO, USA) and counterstaining with hematoxylin.

Alkaline phosphatase activity of osteoblasts was analyzed by a 30-minute incubation in a 0.01% naphtol AS-MX phosphate and 0.24 mg/mL of Fast red TR salt solution (pH 9; all from Sigma) and counterstaining with light green.

Mouse spleen specimens were fixed in 4% buffered formaldehyde and then embedded in paraffin. For histologic study, 3-µm-thick sections were stained with hematoxylin and eosin–saffron and Masson's trichrome. Collagen reaction was noted, and cellular populations of spleen were studied.

Serum markers

Blood sample were obtained from mice on day 36 (puberty), day 48 (end of treatment for protocol 3), and at 1, 3, and 4 months after the end of the treatment by a thoracic puncture at euthanasia. Sera were collected after centrifugation and analyzed for

  • TRACP activity. TRACP activity was measured using the Mouse TRACP Assay (Immunodiagnostic Systems, Ltd., Boldon, UK) according to the manufacturer's instructions. Assay specificity is determined by the color intensity of TRACP kinetic activity. At the end of the assay, the absorbance was determined in each sample by reading in a multiwall plate reader (Wallac VICTOR, Perkin Elmer, Courtaboeuf, France).

  • Osteocalcin activity. Osteocalcin activity was measured using the Mouse Osteocalcin EIA Assay (Demeditec Diagnostics GmbH, Kiel, Germany), which recognizes carboxylated and decarboxylated mouse osteocalcin, according to the manufacturer's instructions. The reaction was stopped and the absorbance of the samples was determined by reading in a multiwall plate reader.

Case report

We report the case of a young male patient (11.5 years old at diagnosis) treated in the French OS2006 protocol for a localized femoral inferior osteosarcoma. Randomized with Zometa, he received 10 intravenous injections of 50 µg/kg Zometa (one every month) in combination with chemotherapy (high-dose methotrexate alternating with etoposide-ifosfamide courses).

Statistics analysis

The Tukey/Bonferroni test was used for comparison of specific bone volume in µCT analysis. Statistical evaluation of bone length and body mass data was performed by Student's t test. Results are given as mean ± SD, and results with p < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Tibial length and body mass

In the first protocol corresponding to high doses of ZOL (0.1 mg/kg twice a week ×8 from days 7 to 28), significant differences in body mass were observed at the end of the treatment (day 28) and up to day 56. For example, differences of 7 and 18 g, respectively, were seen for females treated with ZOL and controls 11 days after the end of treatment (Fig. 2A). This difference diminished with time after the end of the treatment: 14 and 21 g, respectively, for ZOL group versus controls on day 63, corresponding to 1 month after the end of treatment.

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Figure 2. Effect of zoledronic acid (ZOL) on body mass (AC) and tibial length (DF) administered following protocol 1 (0.1 mg/kg, days 7–28, 8-fold: A, D), protocol 2 (0.05 mg/kg, days 14–35, 10-fold: B, E), and protocol 3 (0.05 mg/kg, days 28–46, 10-fold: C, F).

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However, when pediatric doses were used (0.05 mg/kg, protocols 2 and 3), the differences between the two groups were less significant. For example, the mean body masses of females on day 61 (1 month after the end of treatment for protocol 2) were, respectively, 17.5 and 20 g (p < 0.01) for the ZOL and control groups (Fig. 2B). Concerning the third protocol, the mean body mass on day 74 (corresponding to 3 months after the end of treatment) was similar in control and ZOL groups (eg, 20.66 and 20 g, respectively, for females treated with ZOL and PBS; Fig. 2C).

Tibial length was measured for each protocol throughout the treatments by radiographic analysis or by µCT. For the protocol 1, corresponding to a ZOL adult dosing regimen, a significant difference in tibial length was calculated on day 28 (end of treatment): 6.7 mm versus 10.6 mm, respectively, for ZOL and control mice (−27%, p < 0.01). This difference persisted even after the end of treatment. Radiographs of the tibias clearly confirm this effect, with shorter long bones and radiodense lines in the metaphyseal region of tibias and femurs (Fig. 2D). Concerning the second protocol, corresponding to pediatric dosing regimen, a significant difference in tibial length was measured 14 days and 1 month after the end of treatment, but the two values were no longer significantly different 2 months after the end of treatment, revealing a transient inhibitory effect of ZOL on bone length at these doses (Fig. 2E). In protocol 3, the differences were even less significant, reaching between 4% and 6.3% of the difference throughout and after treatment whatever the mouse strain and even 4 months after the end of treatment (Fig. 2F).

Bone microarchitecture

To better analyze the differences in bone microarchitecture observed by radiography, µCT was performed throughout treatment on live animals and at the time of killing. It clearly appeared that ZOL injection lead to the formation of a large ossified band at the metaphyseal area of long bones. In the case of high doses of ZOL (protocol 1), this intense bone formation extended from the growth plate to the metaphysis and a large part of the diaphysis areas, and it was still observed 4 weeks after the end of treatment (day 56; Fig. 3A). When pediatric doses were given for 10 injections, beginning on day 14 (protocol 2) or day 28 (protocol 3; Fig. 3B), a large trabecular ossification area was observed at the metaphysis corresponding to a huge increase in specific bone volume (calculated as 15% of total bone length) from 9.99% in controls to 79.33% in ZOL-treated mice at the end of treatment (day 48; Fig. 3B). When the bone microarchitecture was analyzed up to 4 months after the end of treatment, a shift in this intense bone ossification area was observed in the diaphysis. However, the difference in specific bone volume remains very high: 70.2% for ZOL-treated mice versus 7.22% for controls 4 months after the end of treatment (Fig. 3B).

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Figure 3. Effect of zoledronic acid (ZOL) on specific bone volume analyzed by µCT. ZOL was administered following protocol 1 (A) or protocol 3 (B, C). The specific bone volumes (bone volume/tissue volume) were measured for each volume of interest (VOI) selected as 15% or 50% of the trabecular bone at different times: at the beginning of treatment (day 28), the middle of treatment (day 36), the end of treatment (day 48), and then 1, 2, 3, and 4 months after the end of treatment (B). The effect of ZOL on specific bone volume was compared 3 months after the end of treatment among three different mouse strains (C57BL/6, Swiss, and C3H/HeN) following protocol 3 (C).

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This effect on bone length also was observed in other mouse strains treated with the same protocol 3, but to a lesser extent. Indeed, the difference in bone length was more significant for the C57BL/6 strain (16.8 mm length in controls versus 14.66 mm for ZOL-treated mice 2 months after the end of treatment; Fig. 3C) than for the Swiss strain (18.46 mm for controls versus 17.16 mm in the ZOL-treated group). Finally, no significant change in tibial length could be noticed in C3H/HeN mice (16.86 and 16.43 mm, respectively, for controls and ZOL-treated mice). However, the specific bone volume was always much more elevated in the ZOL-treated mice than in controls, whatever the mouse strain (7.99% in controls versus 72.62% in ZOL-treated mice for the C57Bl/6 strain, 9.95% in controls and 78% in ZOL-treated mice for the Swiss strain, and 15.24% and 68.64%, respectively, for the C3H/HeN strain; Fig. 3C). No atypical fractures were observed under ZOL treatment given at a pediatric dosing regimen, as analyzed by µCT and radiography.

Bone histology

Masson's trichrome staining

Evolution of Masson's trichrome staining corresponding to protocol 3 is presented in Fig. 4A. The radiodense line seen in the metaphyseal region on radiographs and scans of ZOL-treated animals contained unresorbed trabeculae consisting of mixed cartilaginous matrix cores and ossification areas below the physis at the midterm and the end of treatment (respectively, days 36 and 48; Fig. 4A). However, these longitudinal septa retained in the trabeculae were only transiently preserved in the metaphysis. Indeed, this area shifts in the first third of the diaphysis as a consequence of resumption of the functionality of the growth plate (1, 2, and 4 months after the end of treatment), whereas in controls, the trabeculae were subsequently removed from the metaphysis to form the medulla (Fig. 4A). Masson's trichrome staining clearly showed that the medullar space is rapidly recolonized by marrow cells below the physis in ZOL-treated mice 1 month after the end of treatment.

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Figure 4. Histologic analyses of ZOL effects on bone structure by Masson's trichrome staining (A) and osteoclast (B) and alkaline phosphatase activity (C). Tibias were collected from control and ZOL-treated groups (corresponding to protocol 3) at different times as indicated. The tibias were fixed in 4% buffered formaldehyde, decalcified over 4 weeks, dehydrated, and embedded in paraffin as described in the “Materials and Methods.” Then 5-µm-thick sagittal sections were stained with Masson's trichrome (A, magnification ×16). Tartrate-resistant acid phosphatase (TRACP) staining was performed on tibial sections of mice euthanized at the end of the treatment (day 48) or 4 months after the end of treatment to identify the presence of osteoclasts (B, magnification ×50). Alkaline phosphatase (ALP) activity of osteoblasts was analyzed on the same slices (C, magnification ×50).

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TRACP staining

Red-stained TRACP+ cells could be observed all along the growth plate and in trabeculae septa in control mice on day 48 and then only at the physis when endochondral bone formation was diminished (4 months after the end of treatment, corresponding to day 170; Fig. 4B). In ZOL-treated animals, a weak positive staining remained only in the trabeculae septa below the physis at the end of treatment (day 48), but no TRACP staining could be seen at the growth plate nor at the periosteum–cortical bone interface (Fig. 4B). Four months after the end of treatment, TRACP activity is evidenced in the trabeculae area in the diaphysis but also at the physis again. These results reveal that the TRACP activity is decreased by ZOL but remains functional at the growth plate after the end of ZOL treatment.

Alkaline phosphatase staining

Osteoblast activity can be followed by the staining of one of the most characteristic markers: alkaline phosphatase (ALP). The histologic analyses showed comparative staining at the growth plate and in the longitudinal trabecular septa below the physis in both control and ZOL-treated animals at the end of treatment (day 48; Fig. 4C). Four months after the end of treatment, the presence of ALP is retained at the ossification area in the diaphyses of ZOL-treated animals. In controls, osteoblast activity is absent or very low at more than 5 months of age.

Spleen histology

Spleen histology showed no correlation between ZOL treatment and fibrous reaction or architectural disorder (not shown). Elements of the hematopoietic population, especially megakaryocytes, were more abundant on day 48 than later (Table 1). However, these findings were more pronounced for ZOL-treated mice than for controls (Fig. 5).

Table 1. Histologic Determination of Fibrosis by Masson's Trichrome Staining and Hematopoietic Elements (Megakaryocytes) During Zoledronate Treatment According to Protocol 3
TimeTreatmentFibrosisMegakaryocytes
D28 0+
D36CT0+
D36ZOL0+
D36ZOL0+
D48CT0+
D48ZOL+++
D48ZOL+++
+1MCT0+
+1MZOL0+
+1MZOL0++
+3MCT++
+3MZOL+−/+
+3MZOL++
+4MCT++
+4MZOL++
+4MZOL+++
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Figure 5. Histologic evidence of the presence of megakaryocytes in the spleen tissue of mice at the end of ZOL treatment (day 48 in protocol 3). Mouse spleen specimens were fixed in 4% buffered formaldehyde and then embedded in paraffin. Then 3-µm-thick sections were stained with hematoxylin and eosin–saffron. Arrows indicate megakaryocytes. Magnification ×400.

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Biologic markers

In order to complete the study on the effect of ZOL on bone remodeling all along growth, TRACP-5b activity and osteocalcin were measured in the serum of mice treated or not with ZOL following protocol 3. Osteocalcin levels increased both in controls and in ZOL-treated mice from days 28 to 40, then decreased up to day 70, and then remained stable up to more than 5 months (Fig. 6A). ZOL treatment did not significantly modify these variations.

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Figure 6. Effect of ZOL treatment on bone remodeling markers in serum. Blood samples were obtained from mice on day 36 (puberty) and day 48 (end of treatment for protocol 3) and at 1, 3, and 4 months after the end of treatment by a thoracic puncture following euthanasia. Sera were collected after centrifugation and analyzed for osteocalcin (A) and tartrate-resistant acid phosphate 5b (B) activity.

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TRACP-5b activity measured at the systemic level in the serum reflects osteoclast activity. In controls, its concentration dramatically decreased from 20 to 6.5 U/L between days 28 and 48, remaining at a low level thereafter (Fig. 6B). This diminution is consistent with the end of intensive bone remodeling observed along bone growth at mice puberty. ZOL treatment induced a significant decrease of TRACP-5b activity compared with control animals as soon as day 36 (midterm treatment) and up to 3 months after the end of treatment (Fig. 6B).

Case report

The case of a young male patient (11.5 years of age at diagnosis) treated in the OS2006 protocol for a localized femoral inferior osteosarcoma is reported (Fig. 7A). We observed for this patient growing pains with no gain in size during ZOL treatment. Moreover, a dense band appeared just below epiphyseal cartilage at the end of treatment on conventional radiography of the controlateral healthy knee (Fig. 7B). However, clinical development aligned with the growth curve, and growth keep going after the end of treatment with a normal gain in size. Radiologic evaluation showed a gap in the dense band of bone that occurred distal to epiphyseal cartilage (4 months after the end of treatment; Fig. 7C).

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Figure 7. A case report of a young patient (11.5 years of age at diagnosis) treated in the French OS2006 protocol for a localized femoral inferior osteosarcoma. Randomized with Zometa, he received 10 intravenous injections of 50 µg/kg of Zometa (one every month, 4 before and 6 after surgery) in combination with chemotherapy (high-dose methotrexate alternating with etoposide-ifosfamide courses).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

The use of agents that target the tumor microenvironment rather than the tumor itself is one of the most promising innovative approaches to improve survival in osteosarcoma patients.35–37 By targeting the cells that support osteoclastogenesis, bisphosphonates (BPs) are able to inhibit osteoclast activity, thereby reducing bone turnover and increasing bone strength. In addition, BPs show antitumor activity in a number of preclinical and clinical studies.16 In the case of osteosarcoma, BPs inhibit tumor growth and disrupt tumor interactions with the surrounding microenvironment, as demonstrated in preclinical models.38–40 Additional support for the use of BPs in osteosarcoma comes from their potential for reducing fracture risk and prosthesis complications. A recent phase II study conducted at the Memorial Sloan-Kettering Cancer Center in New York City assessed the role of the nitrogen-containing BP pamidronate in the treatment of osteosarcoma. Early indications revealed that BPs can be given safely in the context of osteosarcoma therapy but that metaphyseal changes have been observed frequently in this group of patients. In France, the current protocol for osteosarcoma patients (OS2006) is a multicenter randomized phase III study designed to test the impact of zoledronic acid (Zometa) in association to chemotherapy on progression-free survival in children and adults with osteosarcoma. In this study, one prepubertal osteosarcoma patient treated with ZOL developed metaphyseal sclerotic bands (Fig. 7).

The indications for nitrogen-containing bisphosphonates (eg, pamidronate or zoledronate) in pediatric cancer treatment therefore are increasing with time, and BPs are administrated at higher dosing regimens than those previously given for other skeletal pediatric disorders such as osteogenesis imperfecta (OI), juvenile rheumatoid arthritis, or juvenile idiopathic osteoporosis.29, 30, 41–43 The relevance of using zoledronate, a third-generation N-BP, is linked to its higher potency (two to three orders of magnitude greater than second-generation pamidronate) in bone-resorption assays. In a study published by Pataki and colleagues, zoledronate and pamidronate caused a dose-dependent suppression of cancellous bone turnover and resorption to produce an increase in cancellous bone, but zoledronate was 100 times more potent than pamidronate.44 Therefore, the doses of Zometa used in the French OS2006 protocol are 4 mg intravenously every 4 weeks (×10) for adult patients (>25 years), 0.05 mg/kg every 4 weeks (×10) for patients younger than 18 years (but not exceeding 4 mg as total dose), and 0.05 mg/kg for the first two treatments and then 4 mg for patients between 18 and 25 years of age. The accumulation of high doses of BPs in children and adolescents raised the question of the potential effect on growth. Because these molecules have a high tropism to the bone matrix and accumulate in this tissue,45 intensive doses of bisphosphonates may affect growth properties.

This is why we propose to study the impact of ZOL, a third-generation N-BP on bone properties and growth in mice using three protocols that reproduce both osteosarcoma and Ewing sarcoma dosing regimens and time schedules and taking into account the potential reversibility of such effects. Very few preclinical studies have been reported on the effects of BPs on bone growth. A preclinical study performed previously in rabbits showed that ZOL injected only once or twice at a dose of 0.1 mg/kg caused a transient disruption of physeal morphology and retention of cartilaginous matrix in trabeculae and cortical bone of the metaphysis leading to a minor decrement in tibial bone length at maturity.32 However the dosing regimen used in that study is not comparable with that received by osteosarcoma pediatric patients.

Another study concerning the duration and dose of N-BP administration on bone remodeling in mice showed that high doses of ZOL inhibit both osteoclast and osteoblast functions and in vivo bone remodeling, interfering with bone mechanical properties.46 Two other preclinical studies performed in mice also described an altered bone remodeling during growth in mice treated with BPs, but at lower doses.33, 34

Osteosarcoma arises in adolescents and young adults with a major peak incidence at 18 years of age, whereas Ewing sarcoma is more often observed earlier, in children and adolescents, with a peak incidence at 15 years of age. Therefore, two protocols using pediatric dosing regimens of zoledronic acid (0.05 mg/kg × 10) but beginning at two different times were developed. In patients, the injections are performed every 4 weeks. Although the correspondence between humans and mice is difficult to establish, veterinarians help us to define the best protocol in line with current clinical trials. This is why we chose to administer the ZOL every 2 days in mice, starting on day 14 (protocol 2) or on day 28 (protocol 3). These two different protocols aim at covering the potential alterations induced by ZOL on bone development during childhood and adolescence. In addition, because young adults also received Zometa at adult dosing when they are older than 18 years of age, we included a third protocol with corresponding adult doses (0.1 mg/kg twice a week × 8, protocol 1).

Previous publications have reported the effect of bisphosphonates on growth of children, but in the majority of cases, these studies were done in OI patients who presented with altered bone composition and retarded bone growth from normal height to highly compromised height.29, 47–49 In addition, most of these studies used second-generation BPs, which are less efficient than ZOL, a third-generation N-BP, one of the most potent inhibitors of bone resorption. Therefore, this study is the first that analyzes ZOL effects on bone growth using dosing regimens adapted from oncopediatric protocols. Moreover, the potential reversibility of these effects occurred 1, 2, and 4 months after the end of ZOL treatment, corresponding to more than 10 years in humans.

The results obtained in mice treated from day 7 to day 28 with ZOL, corresponding with adult dosing regimens, showed a high impact on weight and long bone length. This latter effect was caused by a large ossification center in the metaphyseal area that prevents normal bone growth by endochondral ossification. The same trend was observed with dosing regimens adapted from pediatric protocols, but to a lesser extend when considering bone length (protocols 2 and 3) and even not significant for the variation in body mass (protocol 3). Two major parameters can explain this difference: First, the ZOL doses were higher in the first protocol, and it is well known that BPs accumulate in the bone matrix, and second, the mice were treated at a younger age in the first protocol than in the other two protocols. However, when they are compared (protocol 2), the differences in body mass persist even after the end of treatment. Concerning bone length, the difference observed in protocol 3 is never significant (maximum −6%), and the difference observed with protocol 2 is transiently significant, with total reversibility 2 months after the end of treatment (Fig. 2E). Because the second and third protocols are more representative of pediatric dosing regimens, it is reassuring to note that the alterations in body mass and bone length are not significant and are reversible. This increased metaphyseal ossification caused by ZOL reflects its high antiresorptive capability, which is more pronounced at the growth plate during mouse growth because it is the location of endochondral ossification.

These preclinical results obtained in mice can be put together with the case report provided in this article. Indeed, a young patient 11.5 years old at diagnosis showed no gain in size during ZOL treatment, but growth kept going after the end of treatment with a normal gain in size. We cannot demonstrate that the ZOL effect on bone length is reversible, but only that it is transient.

In order to better characterize the mechanisms involved in these phenomena, complementary methodologies were used to analyze bone microarchitecture at the cellular level. The dense metaphyseal bands observed by radiography correspond to high ossification at the growth plates, as revealed by µCT and histologic analyses. Quantification of the specific bone volume calculated to the upper 15% of the tibia revealed a significant elevation of this parameter (8-fold) at the end of the treatment, still persisting even 4 months after the end of treatment (10-fold). This result demonstrated that the elevation in ossification is still visible and measurable long after the end of ZOL treatment but does not preclude the resumption of endochondral ossification from the growth plate. Moreover, osteoclasts and osteoblasts still show intact activity at bone sites to maintain the bone-remodeling process when bone growth started again in young mice.

To explain the intense ossification observed with ZOL treatment, we hypothesize that ZOL disrupted physeal morphology in a transient and dose-dependent manner, these disruptions leading to a change in the form of cartilaginous matrix. Normally, removal of cellular material from continuous, vertical hypertrophic cell stacks flanked by columns of cartilaginous matrix in the physis leaves longitudinal cartilaginous septa that are thinned by resorption before bone deposition on them, as described in rabbits by Smith and colleagues.32 The mechanism of endochondral bone elongation relies on clonal expansion and subsequent hypertrophy of chondrocytes, along with vascular invasion and resorption of the cartilaginous septa at the chondro-osseous junction and influx of cells required for the formation and resorption of primary spongiosa. Furthermore, a continued remodeling of the primary spongiosa is essential for metaphyseal integrity. Preservation of cartilaginous matrix in the metaphysis after BP therapy, as observed in our experiments (Fig. 4), is presumed to occur through an inhibitory action of BP on osteo(chondro)clastic resorbing cell function.50 Moreover, another hypothesis is linked to the antiapoptotic effects of N-BPs on chondrocytes.51

One of the potential consequences of this intense ossification could be a disturbed hematopoiesis owing to less medullar space, as has been reported in mice invalidated for RANKL that showed an osteopetrotic phenotype.52 In our studies, two parameters were analyzed in the spleen tissue: the presence of fibrotic tissue and the quantification of hematopoietic elements, especially megakaryocytes, sign of extramedullar hematopoiesis. Histologic analysis revealed no sign of fibrosis that could be due to ZOL treatment, as has been reported previously in tumor tissue.3 The analysis of hematopoietic elements revealed a slight higher proportion of megakaryocytes observed on day 48 of life in mice that is more pronounced in ZOL-treated mice. This transient augmentation of hematopoiesis in spleen is probably due to the diminution of medullar space in bone.

Biologic markers of bone remodeling also were analyzed during ZOL treatment in young mice based on the variations in osteocalcin and TRACP activity relating, respectively, to bone-formation and bone-resorption processes. An increase in both parameters was observed between days 20 and 30, reflecting intense bone remodeling before and around mouse puberty. In the ZOL-treated group, no significant differences could be quantified for osteocalcin, whereas an significant inhibition of TRACP-5b activity was detected as early as day 35 (7 days after the beginning of ZOL treatment), which persisted up to 3 months after the end of treatment but returned to normal 4 months after.

One of the main limititations of our experimental models in mice is that only 2 days two separated BP injections instead of 4 weeks in humans. During these 2 days in mice, bone remodeling did not have time to compensate for the excess of ossification observed at metaphyseal area caused by the strong inhibitory effect of ZOL on bone resorption. In addition, because BPs accumulate in the bone matrix,45 especially at high doses, this could explain the persistence of medullary bone formation observed long after stopping treatment. However, in patients, the sclerotic bands corresponding to each BP injection seem to be separated, revealing that bone remodeling and bone growth could be preserved. However, the transient stop in growth and the additional bone condensations could slow down the definitive size of young adolescents treated with ZOL over months and aided by the pubertal peak of growth. Further studies are needed to explore patient growth and the definitive size after primitive bone tumor treatment, and analyses should be stratified between pre- or postpubertal patients and ZOL regiment. Such results will be available in ongoing French osteosarcoma protocol OS2006, which randomized conventional chemotherapy alone or combined with ZOL for the treatment of pediatric, adolescents, and young-adults patients.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

This study was supported financially by Novartis Pharma (Rueil-Malmaison, France). All the authors state that they have no other conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We wish to thank Y Allain, C Bailly, and J-M Gaudry from the Experimental Therapy Unit Platform of the IFR26 (Nantes, France) for their technical assistance in animal care. SB and SD contributed equally to this work. This work was supported by a grant from Novartis Pharma (Rueil-Malmaison, France).

Authors' roles: Study design: FR and DH. Study conduct: SB, SD, and JC. Histology analysis: MFH. Microscanner analysis: JC and PG. Clinical interpretation: FG and NC. Drafting manuscript: FR, SB, and JC; Revising manuscript content: NC. Approving final version of manuscript: SB, SD, JC, MFH, PG, FG, NC, DH, and FR.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References