New insights into the pathophysiology and management of bone ...

British Journal of Haematology, 2003, 123, 758–769

Review NEW INSIGHTS INTO THE PATHOPHYSIOLOGY AND MANAGEMENT OF BONE DISEASE IN MULTIPLE MYELOMA

Bone disease is a major feature of multiple myeloma (MM) that is responsible for increased morbidity and mortality. Recent studies have revealed that new molecules such as the receptor activator of nuclear factor-kappa B, its ligand, osteoprotegerin and macrophage inflammatory protein-1 a are implicated in the pathogenesis of myeloma bone disease. These molecules seem to interfere not only with the biology of MM bone destruction, but also with the tumour growth and survival. Currently, bisphosphonates play a major role in the treatment of myeloma bone disease. This review attempts to summarize all the available data for the biology of bone damage in MM and the possible novel targets for developing new drugs that may help in the management of MM bone disease. It also describes the results of all major studies investigating the effects of different types of bisphosphonates on myeloma bone disease, their mode of action and the future implications of their use. Multiple myeloma (MM), which accounts for 10% of malignant haematological diseases, is characterized by the accumulation of malignant plasma cells in the bone marrow. Bone disease is a common clinical feature of MM and correlates with increased morbidity. The manifestation of bone disease includes osteoporosis, hypercalcaemia, lytic lesions and pathological fractures. Vertebral fractures may be associated with spinal cord compression and neurological complications. It has been well documented that MM bone disease is the result of an increased activity of osteoclasts that leads to increased bone resorption in combination with decreased bone formation. The interaction of plasma cells with bone marrow stromal cells (BMSCs) in the bone marrow microenvironment is crucial for the activation of osteoclasts. The use of bisphosphonates, which inhibit bone resorption, for the treatment of MM bone disease has led to an improvement in the quality of life for patients with MM. Several recent studies have provided new insights into the pathogenesis of MM bone disease. Apart from cytokines, such as interleukin-6 (IL-6), IL-1b, IL-11 and the tumour necrosis factors (TNFs), which are known to have osteoclast activating functions, the characterization of newer molecules, such as the receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL), osteoprotegerin (OPG; the decoy receptor of RANKL) and macrophage inflammatory protein-1 alpha (MIP-1a), has helped in further understanding the pathogenesis of MM bone disease and also in developing new therapeutic approaches.

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BIOLOGY OF MM BONE DISEASE Cytokines Osteoclast activating factors (OAFs), first characterized in 1974, were subsequently found to include IL-1b, IL-6, IL11, TNF-a and TNF-b (Mundy et al, 1974). Many other cytokines have also been implicated in the pathogenesis of MM bone disease in vitro, but studies in humans failed to reveal a common model. Increased levels of IL-1b are detected in the supernatant of cultures of freshly isolated myeloma cells and in in vivo models of human MM bone disease (Alsina et al, 1996; Sati et al, 1999). Increased IL-1b mRNA was detected in MM patients compared with those with monoclonal gammopathy of undetermined significance (MGUS) (Donovan et al, 2002), but the protein was not detected in the bone marrow plasma (Choi et al, 2000). In addition, although MM plasma cell supernatants display strong OAF activity, neutralizing anti-IL-1b antibodies fail to completely abolish this activity (Cozzolino et al, 1989). Interleukin-6 is produced mainly by marrow stromal cells after their adherence to myeloma cells and is a growth factor for myeloma cells. It stimulates proliferation and prevents apoptosis of MM cells (Urashima et al, 1996). IL-6 is a potent OAF for human osteoclast precursors and induces bone resorption by human osteoclasts (Reddy et al, 1994). The primary effect of IL-6 on osteoclast formation is to increase the pool of the early osteoclast precursors that in turn differentiate into mature osteoclasts. Therefore, IL-6 stimulatory effects on osteoclast precursors may enhance the effects of bone resorption factors that act at later stages of osteoclast differentiation (de la Mata et al, 1995). IL-6 antisense oligonucleotides inhibit bone resorption by giant cells from human giant cell tumours of the bone (Reddy et al, 1994). However, neither anti-IL-6 nor anti-IL-1b blocking antibody is able to inhibit the osteoclastogenic effects of secreted factors by the cell line ARH-77 in vitro (Alsina et al, 1996), while the administration of chimaeric monoclonal anti-IL6 antibodies in MM had no effect in terms of response (van Zaanen et al, 1998). Serum levels of IL-6 and its receptor (IL-6R) are increased in MM and correlate with disease activity and disease stage (Kyrtsonis et al, 1996; Terpos et al, 2000). In addition, IL-6 and IL-6R serum levels correlate with the duration of disease-free survival (Lauta, 2003). Tumour necrosis factor-a is found at higher levels in the supernatant of plasma cell cultures from MM patients and is

Ó 2003 Blackwell Publishing Ltd

Review capable of stimulating osteoclast formation (Lichtenstein et al, 1989). The effects of TNF-a are mediated by stimulation of the proteolytic breakdown of I-jB, which is the inhibitor of nuclear factor-kappa B (NF-jB), leading to NF-jB activation (Lam et al, 2000). NF-jB translocates into the nucleus where it enhances the transcription of a number of genes, including IL-6, which are involved in promoting bone resorption (Kurokouchi et al, 1998). Interleukin-11 exerts its effect on bone through the RANK/OPG pathway and is produced by both MM cells and BMSCs (Paul et al, 1990). IL-11 induces osteoclastogenesis and subsequently bone resorption. Hepatocyte growth factor (HGF) is involved in angiogenesis, epithelial cell proliferation and osteoclast activation. HGF and its receptor (c-met) is expressed on myeloma cell lines and can be involved in the pathogenesis of bone destruction in MM. HGF up-regulates the expression of IL11 from human osteoclast-like cells, while transforming growth factor-beta 1 (TGF-b1) and IL-1 potentate the effect of HGF on IL-11 secretion. Thus, HGF is speculated to be a possible factor involved in myeloma bone disease (Hjertner et al, 1999). Seidel et al (1998, 2002) have shown that serum HGF levels are elevated in MM patients and predict for poor survival and lack of response to chemotherapy. Chemokines Macrophage inflammatory protein-1a. The MIP-1a is a member of the C–C chemokine family, which is characterized by the absence of an intervening amino acid between the first two of the four cysteine residues that are conserved in the chemokine superfamily. It can interact with three types of chemokine receptors (CCR1, CCR5 and CCR7). MIP1a is primarily associated with cell adhesion and migration, and is chemotactic for monocytes and monocyte-like cells, including osteoclast precursors (Wolpe et al, 1988). Kukita et al (1997) demonstrated that MIP-1a induces osteoclast differentiation in rat marrow cultured on a calcified matrix, while Han et al (2001) have shown that MIP-1a also acts on differentiated osteoclasts in a dose-dependent way, through the receptors CCR1 and CCR5, which are expressed by osteoclasts. Choi et al (2000) were the first to identify MIP-1a as an osteoclast-inducing factor in MM using an expression cloning approach with a human MM cell-derived cDNA expression library. The same group has shown that recombinant human MIP-1a induces osteoclast formation in human bone marrow cultures and that addition of a neutralizing antibody to MIP-1a to human bone marrow cultures treated with freshly isolated marrow plasma from patients with MM blocks MIP-1a-induced osteoclast formation (Choi et al, 2001). MIP-1a has been detected in the supernatant of cultures of bone marrow cells of MM patients and also at the mRNA level in MM cells (Abe et al, 2002; Uneda et al, 2003). MIP-1a levels are elevated in the bone marrow plasma of 56% of MM patients and correlate with the stage and the activity of the disease (Choi et al, 2000), while our group has recently shown that MIP-1a is also elevated in the peripheral blood serum of myeloma patients with severe bone disease (Terpos et al, 2003a).

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In vitro studies have shown that MIP-1a stimulates proliferation, migration and survival of plasma cells (Lentzsch et al, 2003). In addition, when ARH-77 cells are transfected with an antisense construct of MIP-1a and transplanted into severe combined immunodeficient (SCID) mice, the mice live longer and have no radiographically identifiable lytic lesions (Choi et al, 2001). Further evidence of the role of MIP-1a in myeloma bone disease comes from experiments in a myeloma model, in which mice were inoculated with 5TGM1 cells and treated with a monoclonal rat anti-mouse MIP-1a antibody. These mice had a reduction in the paraprotein along with the lytic lesions (Oyajobi et al, 2003). This observation is in accordance with our recent observation, that patients with high MIP-1a serum levels have a poor prognosis (Terpos et al, 2003a). Taken together, these findings suggest an important role of MIP-1a not only in myeloma bone disease but also in myeloma tumour burden. A controversial issue is whether MIP-1a exerts its effect through the RANKL pathway, which is supposed to be the common final mediator of osteoclastogenesis. Han et al (2001) have reported that MIP-1a activity is independent of RANKL, although it enhances the effect of RANKL on osteoclast activation, and that high concentrations of RANK-Fc do not inhibit osteoclast formation induced by MIP-1a. In contrast, Abe et al (2002) reported that MIP-1a induces expression of RANKL by mouse marrow stromal cells. Our group has shown that levels of MIP-1a correlate significantly with those of soluble RANKL (sRANKL) and of markers of bone resorption in MM patients, indirectly supporting the possible role of the RANKL pathway in MIP-1a-induced osteoclast activation (Terpos et al, 2003a). Michigami et al (2000) emphasized the role of cell interactions in the bone marrow microenvironment for the development of myeloma bone disease. They have shown that a murine myeloma cell line binds to stromal cells and induces secretion of osteoclastogenic factors through the binding of very late antigen 4 (VLA-4, a 4b1 intergrin; present on the surface of MM cells) to vascular cell adhesion molecule 1 (VCAM-1), expressed on stromal cells. MIP-1a and MIP-1b can activate intergrins to induce cell adhesion. Therefore, MIP-1a may play a part in a paracrine pathway of inducing adhesion of MM cells to stromal cells through VLA-4/VCAM-1 interactions; thus stimulating osteoclast activation. In addition, high concentrations of MIP-1a produced by myeloma cells attract monocytes/osteoclast progenitors to bone marrow areas infiltrated with myeloma cells and induce their differentiation into mature osteoclasts (Oyajobi et al, 2003). IL-6 might also play its role in myeloma through the activation of MIP-1a; a phenomenon that emphasizes the multiple complex interactions between myeloma and stromal cells (Kuehl & Bergsagel, 2002). Finally, it is of interest that MIP-1a suppresses haemopoiesis, especially erythropoiesis, through its receptor CCR1, which is expressed on erythroid precursors and thus implicated in the pathogenesis of anaemia in MM (Su et al, 1997).

Ó 2003 Blackwell Publishing Ltd, British Journal of Haematology 123: 758–769

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Vascular endothelial growth factor. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has a role in angiogenesis and tumour neovascularization, and has recently been implicated as a mediator for osteoclastogenesis in MM. VEGF is expressed by myeloma cells and it binds to the receptor, VEGFR-1, that is predominantly expressed on osteoclasts. VEGF directly enhances osteoclastic bone resorption and survival of mature osteoclasts (Nakagawa et al, 2000). It can substitute for macrophage colony-stimulating factor (M-CSF) in the induction of osteoclast recruitment in op/op mice (osteopetrotic mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene; Niida et al, 1999). Moreover, Dankbar et al (2000) have shown that VEGF enhances the production of IL-6 from stromal cells, while IL-6 stimulates VEGF expression and secretion in myeloma cell lines and in plasma cells from bone marrow of MM patients. This observation suggests the existence of paracrine interactions between myeloma and marrow stromal cells that are triggered by VEGF and IL-6. RANKL/RANK/OPG system New insights into the pathophysiology of osteoclast differentiation and activation have emerged recently through studies that have characterized three new molecules that belong to the TNF family. RANKL is expressed by activated T cells, marrow stromal cells and osteoblasts and binds to its receptor RANK, which is expressed by osteoclast precursors, chondrocytes and mature osteoclasts (Lacey et al, 1998). The binding of RANKL to RANK promotes osteoclast maturation and activation (Hsu et al, 1999). OPG, which is secreted by stromal cells, is the decoy receptor for RANKL that blocks the RANKL-RANK intreraction and thus inhibits osteoclast differentiation and activation (Simonet et al, 1997). Osteoclasts are multinucleated cells, formed from the fusion of mononuclear progenitors of the monocyte/macrophage family. Osteoclastogenesis requires contact between osteoclast precursors and stromal cells or osteoblasts. These accessory cells express two molecules that are essential to promote osteoclastogenesis: M-CSF and RANKL. It is the balance between the expression of RANKL and OPG that determines the extent of bone resorption. M-CSF expands the pool of osteoclast precursors and RANKL, in turn, stimulates it to commit to osteoclast phenotype by inducing genes that characterize osteoclast differentiation. Stromal cells and osteoblasts are the target cells of most osteoclastogenic factors that exert their effect by enhancing RANKL expression. Such agents include parathyroid hormone (PTH) and 1,15-dihydroxyvitamin D3 (Teitelbaum, 2000). The RANKL is encoded by a single gene at human chromosome 13q14. Alternative splicing of RANKL mRNA allows expression of a type II transmembrane glycoprotein of 45 kDa of either 316 or 270 amino acids, or a soluble ligand of 31 kDa of 243 amino acids. Soluble RANKL can be also released from its membrane-bound state by metalloproteinases, including TNFa-convertase (Kong et al, 1999). OPG is encoded by a single gene on chromosome 8q24 that

leads to the formation of a 110-kDa glycoprotein, which acts as a decoy receptor for RANKL (Morinaga et al, 1998). The importance of RANKL and OPG as positive and negative regulators of osteoclastogenesis, respectively, has become evident from experiments with transgenic mice. Mice that lack either RANKL or RANK or that overexpress OPG develop osteopetrosis because of decreased osteoclast activity (Kong et al, 1999; Kim et al, 2000). Conversely, OPG knockout mice are osteoporotic, develop multiple fractures and have decreased trabecular bone volume and numerous osteoclasts, as OPG cannot inhibit RANKL activity (Bucay et al, 1998). It is noteworthy that mice that are deficient in either RANKL or RANK also exhibit defective lymph node organogenesis and early B-cell development (Kong et al, 1999). Whether the cytokines already characterized as OAFs, exert their effect through the RANKL/OPG pathway is an important issue. It is known that the expression of RANKL is enhanced by glucocorticoids, IL-1b, TNF-a, IL-11, PTH, prostaglandin-E2, vitamin D3 and is decreased by TGF-b (Hofbauer et al, 2001; Sordillo & Pearse, 2003). Expression of OPG is increased by IL-1b and TNF-a, as well as by TGF-b, 17b-oestradiol and CaCl2. Conversely, glucocorticoids, vitamin D3 and PTH reduce OPG production. However, IL-6 seems to support human osteoclast formation by a RANKL-independent mechanism (Hofbauer et al, 2001; Kudo et al, 2003). RANKL/RANK/OPG system in MM. Giuliani et al (2001) have shown that human myeloma cells up-regulate the expression of RANKL and downregulate the expression of OPG at mRNA and protein level in pre-osteoblastic or stromal cells in co-culture systems. RANKL expression at both the mRNA and protein level is increased in bone marrow biopsies from patients with MM compared with those with MGUS (Sezer et al, 2003a). RANKL is expressed by stromal cells, osteoblasts and activated T cells (CD30+) in areas infiltrated by MM (Roux et al, 2002), but the expression of RANKL on myeloma cells remains controversial. Giuliani et al (2001) could not detect RANKL mRNA or the protein by immunohistochemistry in human myeloma cells. Experiments by Pearse et al (2001) also support the notion that RANKL is not expressed by MM cells. However, the number of groups who found RANKL expression of myeloma cells is higher than those who did not (Mitsiades et al, 2001). Croucher et al (2001) found that mouse myeloma cells (5T2MM) express RANKL mRNA and the RANKL protein is found on the cell membrane. Using flow cytometry, Sezer et al (2002) detected a strong expression of RANKL on bone marrow plasma cells in MM patients and have defined the presence of the protein in human myeloma cell lines. Vanderkerken et al (2003a) have demonstrated the expression of RANKL by both 5T2MM and 5T33MM cells at both the mRNA and the protein level. Furthermore, the expression of RANKL on bone marrow plasma cells correlates with osteolytic bone disease in patients with myeloma (Farrugia et al, 2003; Heider et al, 2003).


Review In co-culture models, MM cells failed to induce differentiation of murine bone marrow precursors to osteoclasts, when they were supported by stromal/osteoblast cells from RANKL knockout mice, indicating that these MM cell lines could not produce RANKL even after co-culture with murine stromal cells/osteoblasts. The ability of MM cells to induce osteoclast development was reconstituted, when they were supported with stromal cells from the wild-type mice. The in vivo proof of the role of RANKL on MM bone disease comes from experiments in myeloma murine models, where administration of RANK-Fc prevents myeloma-induced bone destruction (Pearse et al, 2001). Osteoprotegerin expression has been found to be reduced in bone marrow specimens from myeloma patients. In co-culture experiments, as already mentioned, the adhesive interactions of myeloma cells with BMSCs inhibit OPG production both at the mRNA and protein level (Giuliani et al, 2001). Furthermore, myeloma cells decrease OPG availability by intrernalizing it through syndecan. Syndecan-1 (CD-138), a transmembrane proteoglycan with heparan sulphates binds to OPG through the heparinbinding domain of the OPG protein, which is then internalized and degraded within the lysosomal compartment of MM cells (Standal et al, 2002). Thus, in MM, the regulation of OPG at the transcriptional and post-translational level reduces the availability of OPG in the bone marrow microenvironment, leading to reduced inhibition of RANKL and increased osteoclast differentiation and activation. Indeed, when serum levels of OPG were evaluated in MM patients, they were found to be decreased. OPG serum levels were lower by 18% in 225 patients with MM (Seidel et al, 2001), and by 29% in 34 patients (Lipton et al, 2002), when compared with controls. OPG levels in bone marrow plasma were lower by 27% in 33 patients in comparison with controls (Standal et al, 2002). In these studies, there was a strong inverse correlation between OPG serum levels and the presence of osteolytic lesions. Our group has shown that serum levels of OPG were reduced, while serum levels of sRANKL were increased in 121 MM patients compared with controls; thus the ratio of sRANKL/OPG was increased in MM. sRANKL/OPG ratio correlated with the extent of bone disease and with makers of bone resorption (including tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), and N-telopeptide of collagen type-I (NTX), suggesting the importance of the RANKL/OPG pathway in the pathogenesis of MM bone disease in humans (Terpos et al, 2003b). All the above interactions between myeloma and marrow stromal cells that lead to increased osteoclast activation and increased bone resorption are depicted in Fig 1. MANAGEMENT OF MYELOMA BONE DISEASE Bisphosphonates Bisphosphonates are potent inhibitors of osteoclast activity and function, playing a major role in the management of myeloma bone disease so far. Their core structure is formed by two phosphonate groups attached to a single carbon atom (P–C–P structure), which is similar to endogenous

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pyrophosphate, but with carbon replacing the central oxygen. In contrast to pyrophosphates, bisphosphonates are very stable. First generation bisphosphonates, such as etidronate and clodronate, are metabolically incorporated into non-hydrolyzable analogues of ATP that inhibit ATP-dependent intracellular enzymes. Second generation bisphosphonates (aminobisphosphonates), including pamidronate, ibandronate, alendronate and risendronate, inhibit the mevalonate pathway leading to the post-translational modification of guanidine triphosphatases (GTPases) (Fleisch, 1998). All bisphosphonates have a high affinity for bone minerals and are preferentially delivered to sites of increased bone resorption or formation (Body, 1998). Bisphosphonates inhibit osteoclastic bone resorption by inhibiting osteoclastic recruitment and maturation, preventing the development of monocytes into osteoclasts, inducing osteoclast apoptosis and interrupting their attachment to the bone. In view of the accumulation of the bisphosphonates in bone, it is of great clinical interest that the inhibition of bone resorption reaches a certain steady level even when the compounds are given continuously, suggesting that, at the therapeutic dosage, there is no danger of a continuous decrease in bone turnover in the long run (Fleisch, 1998). Although all bisphosphonates have similar physico-chemical properties, their anti-resorbing activities differ substantially. For example, pamidronate and alendronate are approximately 100- and 700-fold more potent than etidronate, both in vitro and in vivo, while newer bisphosphonates, such as ibandronate and zoledronic acid show 10 000- to 100 000-fold greater potency than etidronate in vitro (Lin, 1996). In addition to the well-proven anti-resorptive efficacy of bisphosphonates, anti-myeloma activity has also been suggested, mainly from in vitro studies in myeloma cell lines and also from preclinical in vivo models (Dhodapkar et al, 1998; Yaccoby et al, 2002). Pamidronate has been found to be more effective than clodronate, while zoledronic acid was more effective than pamidronate in terms of inducing myeloma cell apoptosis in myeloma cell lines (Shipman et al, 1997; Tassone et al, 2000). Possible mechanisms explaining the anti-myeloma effect of bisphosphonates include the reduction in IL-6 secretion by BMSCs or the expansion of c/d T cells with possible anti-MM activity (Derenne et al, 1999; Kunzmann et al, 2000). Furthermore, it has been reported that zoledronic acid prolongs survival in myeloma animal models (Croucher et al, 2003a), while pamidronate has either direct or indirect anti-tumour effect in patients with MM (Gordon et al, 2002). Ibandronate reduces the development of osteolytic lesions in murine myeloma models, but no anti-myeloma effect of ibandronate has been established to date (Dallas et al, 1999; Shipman et al, 2000; Cruz et al, 2001; Croucher et al, 2003b). Due to their strong anti-resorptive effect and to their possible anti-myeloma effect, several studies have been carried out on the role of bisphosphonates in patients with MM. Etidronate was found to be ineffective in two placebocontrolled studies (Belch et al, 1991; Daragon et al, 1993).


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Review OPG

RANKL

Myeloma cell α4β1 integrin VCA M-1

(-) OPG

Osteoblast/BMSC IL-6 RANKL

RANKL

MIP 1-α HGF

RANK IL1β, IL11, TNFs, bFGF

M-CSF

Osteoclast progenltor

TRACP-5b

Activated osteoclast

NTX

Bone matrlx

Fig 1. The adherence of MM cells to bone marrow stromal cells (BMSC)/osteoblasts enhances the production of RANKL, M-CSF and other cytokines with OAF activity (IL-6, IL-1b, TNFs, bFGF, IGFs), while it suppresses the production of OPG (the decoy receptor of RANKL). The above cytokines also modify the bone marrow microenvironment, up-regulating the RANKL expression and secretion by both BMSC and osteoblasts. Furthermore, myeloma cells produce MIP-1a, HGF and VEGF, which enhance the proliferation and differentiation of osteoclast precursors, while it seems that myeloma cells express both transmembrane and soluble RANKL. OPG binds both surface and soluble RANKL, inhibiting osteoclast development and bone resorption. Syndecan 1 (CD138) expressed on the surface of, and secreted from, the myeloma cells can bind soluble OPG, thus preventing its inhibitory effect on RANKL function. Therefore, the ratio of RANKL/OPG is increased, leading to osteoclast differentiation, proliferation and activation, and to increased bone resorption, as reflected by the increased levels of bone resorption markers (NTX, TRACP-5b).

Clodronate. To date, two major, placebo-controlled, randomized trials have been performed in MM. Lahtinen et al (1992) have reported a trial of 350 newly diagnosed patients who were treated with oral melphalan and prednisolone, and were randomized to receive either clodronate (2400 mg/d) or placebo for 2 years. The major finding was the reduction in the development of new osteolytic lesions by 50% in the clodronate arm. The benefits of clodronate were comparable in patients who had lytic lesions and in those who had no lytic lesions at baseline (Laakso et al, 1994). The Medical Research Council in the UK has performed the largest trial on clodronate in MM (McCloskey et al, 1998). In addition to their chemotherapy, 536 newly diagnosed patients were randomized to receive either clodronate (1600 mg/d) or placebo for a median follow-up of 8Æ6 years. Although there was no difference in overall survival between the two groups, clodronate patients who did not have vertebral fractures at baseline seem to have a survival advantage (59 months vs. 37 months) (McCloskey et al, 2001). After 1 year of follow-up, non-vertebral fractures were 13Æ2% in the placebo group and 6Æ8% in the clodronate group (P ¼ 0Æ036). Vertebral fractures were also reduced in the clodronate group (38% vs. 55% in the

placebo group; P < 0Æ001). The time to first non-vertebral fractures or to severe hypercalcaemia was longer in the clodronate group (P < 0Æ021). At 2 years, the patients who received clodronate had a better performance status and less back pain than patients treated with placebo. Less than 10% of patients needed radiotherapy and there was no difference in survival between the two groups (McCloskey et al, 1998). Furthermore, clodronate has been proven to reduce bone pain, pathological fractures, and biochemical markers of bone resorption in other small trials (Merlini et al, 1990; Clemens et al, 1993). Pamidronate. Two, double-blind, placebo-controlled trials using pamidronate have been performed to date in patients with MM; one using oral administration and one with an intravenous formulation. Brincker et al (1998) carried out a trial in which 304 patients were randomized to receive either oral pamidronate at a dose of 300 mg/d, or placebo, in addition to conventional treatment. The authors found no reduction in skeletal-related events (SREs), defined as bone fractures, related surgery, vertebral collapse, or increase in number and/or size of bone lesions. However, patients treated with oral pamidronate experienced fewer episodes of severe pain and decreased reduction in body height. The overall


Review negative result of this study was attributed to the very low absorption of orally administered bisphosphonates. In the second trial, 392 patients with advanced disease and at least one lytic lesion were randomized to placebo or pamidronate, given intravenously at a dose of 90 mg, as a 4-h infusion, every 4 weeks, for 21 cycles (Berenson et al, 1996). The first analysis was performed after 9 months of treatment. The mean number of SREs per year was reduced from 2Æ1 in the placebo group to 1Æ1 in the pamidronate group at 9 months (P ¼ 0Æ0006), and from 2Æ2/year in the placebo group to 1Æ3/year in the pamidronate group after 21 months of treatment (P ¼ 0Æ008). Furthermore, the median time to the first skeletal event was 10 months in the placebo group and 21 months in the pamidronate group (P < 0Æ001). At 21 months, 51% of patients in the placebo group versus 38% in the pamidronate group had an SRE (P ¼ 0Æ015). The proportion of patients developing a pathological fracture or requiring bone radiation was lower in the pamidronate group. Pain scores and quality of life were significantly improved in the pamidronate group. Although there was no difference in terms of survival between the two treatment groups, this study identified a subgroup of patients, who had received two or more previous anti-myeloma regimens, in which pamidronate administration was associated with prolongation of survival compared with controls (21 months vs. 14 months respectively; P ¼ 0Æ041) (Berenson et al, 1998). Our group has also shown, in 62 newly diagnosed myeloma patients, that intravenous pamidronate, at a dose of 90 mg/month, may have a synergistic action with chemotherapy in reducing not only bone resorption, but also myeloma-related pain and markers of disease activity in patients with MM (Terpos et al, 2000). The Cochrane Myeloma Review Group has reported a meta-analysis based on 11 trials that involved 2183 assessable patients. This review concluded that both intravenous pamidronate and clodronate reduce the incidence of hypercalcaemia, the pain index and the number of vertebral fractures, while they confer no benefit in terms of reducing the number of nonvertebral fractures (Djulbegovic et al, 2002). However, another investigation of the role of oral bisphosphonates in MM and breast cancer concluded that oral bisphosphonates do not seem to be as effective as those administered intravenously (Major et al, 2000). A randomized trial comparing these two bisphosphonates is necessary before final conclusions can be drawn. Zoledronic acid. Two randomized trials have shown that zoledronic acid can be given safely at a dose of 4 mg, intravenously over 15 min, every month, producing similar effects as 90 mg of pamidronate. Berenson et al (2001) compared the effects of zoledronic acid and pamidronate in a phase II randomized trial. One hundred and seventy-two patients with breast cancer and 108 patients with MM and lytic lesions were randomized to 9 monthly infusions of 0Æ4, 2Æ0 or 4Æ0 mg of zoledronic acid in a 5-min infusion, or to 90 mg of pamidronate as a 2-h infusion. Zoledronic acid, at doses of 2Æ0 and 4Æ0 mg, and pamidronate at a dose of 90 mg, each significantly reduced the need for radiotherapy to bone in contrast to 0Æ4 mg zoledronic acid. SREs also

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occurred less frequently in patients treated with 2Æ0 or 4Æ0 mg zoledronic acid or pamidronate than with 0Æ4 mg zoledronic acid. Increases in lumbar spine bone mineral density (BMD) and decreases in bone resorption markers were observed in all treatment groups. Skeletal pain, fatigue, nausea, vomiting and headache were the most commonly reported adverse events and were similar in frequency in zoledronic acid and pamidronate groups. This phase II trial failed to show any superiority of zoledronic acid compared with pamidronate in terms of SREs, in contrast to a large phase III study showing a superior effect of zoledronic acid at 4Æ0 or 8Æ0 mg, over pamidronate for the treatment of hypercalcaemia of malignancy (Major et al, 2001). Therefore, a large phase III, randomized, double-blind, study was performed to compare the effects of zoledronic acid and pamidronate on SREs in patients with breast cancer and myeloma (Rosen et al, 2001). A total of 1130 patients with breast cancer and skeletal metastases and 513 patients with MM who had at least one lytic lesion were randomized to receive 4Æ0 or 8Æ0 mg of zoledronic acid, or 90 mg of pamidronate every 3–4 weeks for 12 months. The 8Æ0 mg arm of zoledronic acid was subsequently stopped due to the risk of renal impairment. The median time to the first SRE was approximately 1 year in each treatment group. The skeletal morbidity rate was slightly lower in patients treated with zoledronic acid than in those treated with pamidronate and zoledronic acid (4 mg) significantly decreased the incidence and event rate for bone radiotherapy. Pain scores were decreased in all treatment groups and were associated with stable or decreased analgesic use. NTX showed better normalization in patients treated with 4Æ0 mg of zoledronic acid compared with pamidronate (P ¼ 0Æ015), and that was the only reported difference between the treatment groups. Zoledronic acid at a dose of 4 mg and pamidronate were equally well tolerated. Ibandronate. Ibandronate has been used to treat the hypercalcaemia of malignancy. It has been proven to be safe and well-tolerated at a dose of 2Æ0, 4Æ0 or 6Æ0 mg given intravenously, as a 2-h infusion (Ralston et al, 1997). However, a recent randomized, double-blind, placebo-controlled trial failed to show any effect of 2Æ0 mg of ibandronate on reducing bone morbidity or on prolonging survival in MM (Menssen et al, 2002). Explorative post hoc analyses revealed that ibandronate patients with strongly suppressed bone-turnover markers developed significantly less bone morbidity. Our group compared the effect of ibandronate and pamidronate on bone remodelling and disease activity in MM (Terpos et al, 2003c). Patients with stage II or III MM were randomly assigned to receive either pamidronate 90 mg (23 patients) or ibandronate 4 mg (21 patients) as a monthly intravenous infusion in addition to conventional chemotherapy. Skeletal events, such as pathological fractures, hypercalcaemia and bone radiotherapy were analysed. In both groups, the combination of chemotherapy with either pamidronate or ibandronate produced a reduction in bone resorption and tumour burden showing no


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effect on bone formation. However, there was a greater reduction in biochemical markers of bone resorption, IL-6 and b2-microglobulin in the pamidronate group than in the ibandronate group; a reduction that was sustained throughout the follow-up of this study. There was no difference in skeletal events during the relatively short period of followup. As ibandronate was effective in reducing bone resorption and IL-6 levels, but inferior to pamidronate in the dose used in our trial, a higher dose (6 or 8 mg) of ibandronate may be more efficacious. However, this remains to be evaluated in randomized trials. What is the optimal duration of bisphosphonates in myeloma patients? This question has yet to be answered because this issue has never been the subject of any clinical trial. However, due to the benefit of bisphosphonates on performance status, quality of life and possibly on survival in a subset of patients, the clinician has to decide the optimal duration taking into account the potential palliative benefits of clodronate, pamidronate or zoledronic acid and the adverse events that may be manifested. The time of initiating the bisphosphonates treatment is also controversial. All patients with MM who have lytic lesions need to start bisphosphonates therapy. The American Society of Clinical Oncology has suggested that myeloma patients with osteopenia alone may be treated with bisphosphonates, but there is no such recommendation for patients with solitary plasmacytoma or smoldering/indolent myeloma without documented lytic bone disease (Berenson et al, 2002; Sezer et al, 2003b). Monitoring bisphosphonates treatment in MM. Biochemical markers of bone turnover have been used in patients with MM in order to identify subsets of patients who are most at risk of bone complications, to identify patients who will benefit the most from bisphosphonate treatment and also for predicting progression of bone disease. A variety of markers of bone resorption [NTX, C-telopeptide of collagen type-I (ICTP), TRACP-5b, pyridinoline and deoxypiridinoline) and markers of bone formation [bone alkaline phosphatase (bALP), osteocalcin (OC), and procollagen type-I N- or Cpropeptide (PINP and PICP respectively)] have been studied. Both ICTP and NTX have shown a dramatic decrease after pamidronate or zoledronic acid administration, confirming the strong anti-resorptive activity of these agents (Terpos et al, 2000, 2001; Berenson et al, 2001). A recent study has shown that high levels of ICTP and NTX correlated with an increased risk for early progression of bone lesions during standard melphalan-prednisolone treatment in MM. This study suggests that ICTP and NTX are clinically useful for identifying those patients with an increased risk of early progression of bone disease (Abildgaard et al, 2003). Furthermore, our group has shown that TRACP-5b, a novel marker of bone resorption that is produced only by activated osteoclasts, is increased in MM patients, correlates with the extent of bone disease, is reduced during pamidronate administration and possibly has a predictive value in bone-disease and tumour progression (Terpos et al, 2003d). Biochemical markers of bone resorption have also been found to have prognostic value in MM, while we have recently shown that serum markers of osteoclast function,

including the ratio sRANKL/OPG, predict for survival in MM (Abildgaard et al, 1997; Fonseca et al, 2000; Terpos et al, 2003b). In addition, these markers seem to be normalized after high-dose chemotherapy with autologous stem cell support (Clark et al, 2000; Terpos et al, 2002). Although the results of these studies are interesting, further trials are needed to establish the predictive value of these markers before introducing them into routine use. RANKL/OPG system as a target for the management of myeloma bone disease The first experiments modulating the RANKL/OPG system were by Pearse et al (2001) who used the SCID ARH-77 xenograft and the SCID-huMM, in which the human myeloma cell line (ARH-77) and primary human bone marrow cells, respectively, were injected into mice. Both groups of animals were treated with intravenous injections of RANK-Fc, a fusion protein of the murine RANK with the human IgG Fc region. The treatment results in markedly reduced bone resorption and absence of skeletal destruction. In the SCID-huMM model the effect on bone disease is accompanied by reduction in serum paraprotein and reduction in tumour burden. Croucher et al (2001) have demonstrated that injection of 5T2MM myeloma cells in C57BL/KalwRij mice results in the development of bone disease. Treatment with recombinant OPG prevents the development of lytic bone lesions, inhibits osteoclast formation and promotes an increase in femoral, tibial and vertebral BMD. The same effect of OPG has been observed in the SCID ARH-77 xenograft model in which they used ex vivo gene transfer of the OPG-Fc gene using a lentiviral vector. The overexpression of the OPG results in a lower incidence of osteolytic lesions and in longer survival (Sezer et al, 2003a). Vanderkerken et al (2003b) have reported that treatment of mice injected with 5T33MM cells with OPG-Fc causes a significant decrease in osteoclast number, along with the reduction in serum paraprotein and significant increase in time to morbidity. Body et al (2003) first attempted to disrupt the RANKL/ RANK interaction in their trial of MM patients, who were randomized to receive a single dose of either AMGN-0007 (recombinant OPG), subcutaneously, or 90 mg of pamidronate, intravenously, and were followed for 56 d. AMGN0007 caused a rapid and sustained dose-dependent decrease in NTX that was comparable with that observed with pamidronate. The drug has not had any major adverse effects. A longer follow-up period is needed to evaluate the effect of AMGN-0007 on bone disease in MM patients. However, these results warrant further clinical trials targeting RANKL/OPG. It is of interest that in most of the above studies, the normalization of the RANKL/OPG ratio, either by inhibiting RANKL or by administering OPG, resulted not only in the reduction in bone disease but also in tumour burden as reflected by the reduction in paraprotein levels and the prolongation of survival of mice in the animal myeloma models. Our group has recently shown that the RANKL/ OPG ratio correlates with survival in patients with MM.


Review Those patients with a ratio below 1 had a 5-year probability of survival of 89%, while no patient with a ratio above 3 survived for more than 4 years (Terpos et al, 2003b). These observations reflect the importance of the interaction of plasma cells with BMSCs. MM cells enhance the production of OAFs from stromal cells, which in turn promote the expression and secretion of factors that promote accumulation and survival of MM cells. Through an unknown mechanism, OPG has also been shown to participate in this paracrine loop and, apart from its role on osteoclastogenesis, seems to function as a survival factor for MM cells as well (Shipman & Croucher, 2003). CONCLUSIONS Bone disease remains a major problem in the management of patients with MM. Bisphosphonates constitute a valuable group of agents for the treatment of myeloma bone disease having a strong anti-resorptive activity and possibly an anti-tumour effect. Oral clodronate, intravenous pamidronate or intravenous zoledronic acid should be used in myeloma patients with osteolytic bone disease. However, many important issues, such as the most effective bisphosphonate, the time of initiation, the duration of treatment and the use of markers to select high-risk patients, have not yet been clearly clarified. Therefore, additional studies focusing on these issues are required. Furthermore, the emergence of new molecules that are involved in the pathogenesis of MM bone disease (RANKL, OPG, MIP-1a, etc.) may enable the development of new drugs, which may not only improve MM bone disease but also reduce myeloma tumour burden. Department of Haematology, Evangelos Terpos Faculty of Medicine, Marianna Politou Imperial College London, and Amin Rahemtulla Hammersmith Hospital, London, UK Correspondence: Amin Rahemtulla, Department of Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: [email protected] REFERENCES Abe, M., Hiura, K., Wilde, J., Moriyama, K., Hashimoto, T., Ozaki, S., Wakatsuki, S., Kosaka, M., Kido, S., Inoue, D. & Matsumoto, T. (2002) Role for macrophage inflammatory protein (MIP)1alpha and MIP-1beta in the development of osteolytic lesions in multiple myeloma. Blood, 100, 2195–2202. Abildgaard, N., Bentzen, S.M., Nielsen, J.L. & Heickendorff, L. (1997) Serum markers of bone metabolism in multiple myeloma: prognostic value of the carboxy-terminal telopeptide of type I collagen (ICTP). Nordic Myeloma Study Group (NMSG). British Journal of Haematology, 96, 103–110. Abildgaard, N., Brixen, K., Kristensen, J.E., Eriksen, E.F., Nielsen, J.L. & Heickendorff, L. (2003) Comparison of five biochemical

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Keywords: multiple myeloma, bone disease, bisphosphonates, receptor activator of nuclear factor-kappa B ligand, osteoprotegerin.
