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Leukemia Research 31 (2007) 129–138

Invited review

Myeloma bone disease: Pathogenetic mechanisms andclinical assessment

Franco Silvestris ∗, Lucia Lombardi, Monica De Matteo,Arianna Bruno, Franco Dammacco

Department of Internal Medicine and Clinical Oncology (DIMO), University of Bari, P.za Giulio Cesare, 11-70124 Bari, Italy

Received 30 March 2006; accepted 15 April 2006Available online 9 June 2006

bstract

Bone disease in multiple myeloma (MM) leads to progressive devastation of the skeleton and is the most severe cause of morbidity. Itsathogenetic mechanisms are not fully defined, though the current evidence points to hyperactivation of osteoclasts (OC) in presence of aajor defect of bone repairing in erosion sites due to osteoblast (OB) impairment. Bone resorption, however, is promoted by early OB, namely

tromal cells that respond to chronic stimulation by myeloma cells by enhancing marrow levels of RANKL and other osteoclastogenic factorsnd thus accelerating the maturation of OC progenitors. In myeloma bone disease (MBD), OBs are systematically defeated by a numberf inhibiting effects induced by the malignant clone within the marrow microenvironment. Thus, MBD primarily affects the OB lineage,articularly in overt MM, where serum markers of osteoblastogenesis, such as osteocalcin and osteoprotegerin, are extremely low in contrastith their slight increase in inactive MM. These markers, in association with others of bone turnover (RANKL, MIP-1�, type I collagen

elopeptides such as NTX and CTX) may be used in the clinical assessment of MBD as well as to monitor the efficacy of bisphosphonate inelaying the progressive skeletal destruction.

2006 Elsevier Ltd. All rights reserved.

eywords: Bisphosphonates; Bone marrow microenvironment; Multiple myeloma; Osteoblasts; Osteoclasts; Osteoprotegerin; RANKL

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Multiple myeloma (MM) is a B cell neoplasm charac-erized by expansion within the bone marrow of a malig-ant plasma cell clone which leads to a destructive boneisease, namely the formation of typical osteolytic lesions.rogressive skeletal impairment is the most severe cause oforbidity and results in prominent fractures occurring either

pontaneously or following trivial injuries, spinal cord com-ression, intractable bone pain and extreme disability. Theechanisms involved in the pathogenesis of myeloma bone

isease (MBD) are not fully elucidated, though the preva-ent opinion is that malignant plasma cells exert a major

steoclastogenic effect by promoting the recruitment, differ-ntiation and activation of osteoclast (OC) progenitors withinhe bone marrow following their interaction with the stro-

∗ Corresponding author. Tel.: +39 080 5478 771; fax: +39 080 5478 831.E-mail address: f.silvestris@dimo.uniba.it (F. Silvestris).

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145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2006.04.014

al cells [1]. These cells, which physiologically differentiatento osteoblasts (OB) in the bone marrow, produce severalsteoclastogenic factors and remain deregulated under thenfluence of the malignant plasma cell clone, and this resultsn persistently elevated marrow concentrations of those fac-ors.

Enhanced bone resorption, however, is not accompaniedy concurrent enhancement of OB function to replace theone loss. This peculiar feature of the MBD is sustainedy the extreme paucity of osteocytes or newly formed boneithin or near the erosion sites. These, indeed, rarely heal

ven in patients achieving chemotherapy-induced completeemission [2] where bone remodeling is unresponsive to com-

on osteotrophic factors, such as ionized calcium, calciferol

nd/or calcitonin. Conversely, OC hyperactivity can be effi-iently down-modulated or even normalized by bisphospho-ates, alone or associated with conventional chemotherapy.

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primary OB defect is thus provoked by cell-to-cell con-act with malignant plasma cells since responsible for bothncontrolled release of osteoclastogenic factors and persis-ent inability to replace the osteolytic lesions with newlyormed bone [3].

This paper reviews the main pathogenetic mechanisms andhe clinical assessment of MBD in the light of our own studiesnd the literature.

. Pathophysiology of MBD

Several studies have focused on the molecular events con-ected with the development of MBD, with particular regardo enhanced OC function and depressed OB activity.

.1. Hyperactive osteoclastogenesis

It is now becoming increasingly clear that the produc-ion of locally acting osteoclastogenic and resorptive factorsy both OBs and stromal cells is dependent on a numberf molecular interactions between each other and the malig-ant plasma cells. This forms a neoplastic unit that releasesowerful inflammatory and erosive cytokines, namely inter-eukin (IL)-1�, IL-3, IL-6 (which acts as survival factor for

yeloma cells), IL-11, tumor necrosis factor (TNF)-� and -

, parathyroid hormone-related protein (PTHrP), hepatocyterowth factor, basic fibroblast growth factor (bFGF), metal-oproteases and macrophage inflammatory protein (MIP)-1�4]. An incomplete list of these inflammatory factors and

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able 1ncomplete list of marrow soluble factors driving osteoclastogenesis in myeloma bo

Cellular origin

ytokinesIL-I� Myeloma cellsIL-3 Marrow T cellsIL-6 Stromal and myeloma cellsIL-11 Stromal and myeloma cellsTNF-� Myeloma cellsTNF-� Myeloma cellsHGF Myeloma cells

hemokinesMIP-l� Macrophages and myeloma cellsVEGF Myeloma cells

steoclastogenic factorsRANKL Stromal and myeloma cellsM-CSF Stromal cells

ther factorsMMP-7 Myeloma cellsMMP-13 OCMMP-14 OCPTHrP Myeloma cellsbFGF Stromal cells

IL) interleukin; (TNF) tumor necrosis factor; (HGF) hepatocyte growth factor; (MIactor; (RANKL) ligand of the receptor activating the nuclear factor kB; (M-CSF) marathyroid hormone-related protein; (bFGF) basic fibroblast growth factor; (OC) o

earch 31 (2007) 129–138

heir cellular origin within the neoplastic unit is reported inable 1.

The primary subversion in cytokine production by bothyeloma and stromal cells is induced by a number of recip-

ocal interactions between them. After their extravasation,ntegrins such as very late antigen (VLA)-4, �v�3 and �4�1xpressed by malignant plasma cells bind to vascular celldhesion molecule (VCAM)-1 on stromal cells, resulting introng mutual adhesion subsequent to the reciprocal attrac-ion exerted by several chemokines such as MIP-1� and-1�.he transcriptional activity of stromal cells is also impaired,ith consequent derangement of their production of regu-

atory factors within the marrow. In addition, maturation ofarly OBs is prevented because of their enhanced apoptosis,hereas deregulated stromal cells release increased amountsf two major osteoclastogenic factors, namely the ligand ofhe receptor activator of nuclear factor kB (RANKL) andhe macrophage-colony stimulating factor (M-CSF). Fig. 1ummarizes the sequences of events preceding the OC hyper-ctivity in MBD.

High marrow levels of these cytokines result in bothecruitment and differentiation of OC progenitors, namelyesident macrophages, leading to their proliferation, cellusion, inhibition of apoptosis and functional hyperactivity. Inddition, osteoprotegerin (OPG), a soluble decoy receptor ofANKL physiologically released by marrow stromal cells to

ounterbalance excessive osteoclastogenesis, is significantlyuppressed in MBD patients [5]. The reason for this is notlear, though functional deregulation of stromal cells follow-ng their assimilation within the neoplastic unit may lead to

ne disease

Biological function

Inflammation inductionStromal cell activationMyeloma cell growthOC activation through RANKL/OPGPathwayApoptosis activationApoptosis activation inflammatory induction

Chemotaxis activation in OC precursorsOC activation, IL-6 induction by stromal cells

Major OC activationOC differentiation induction

Protein digestionBone matrix degradationBone matrix degradationStromal cell stimulationStromal cell proliferation

P) macrophage inflammatory protein; (VEGF) vascular endothelial growthacrophage-colony stimulating factor; (MMP) metalloproteinase; (PTHrP)

steoclast.

F. Silvestris et al. / Leukemia Research 31 (2007) 129–138 131

Fig. 1. Mechanisms of osteoclastogenesis in MBD. Extravasated malignant plasma cells adhere to stromal cells by both �4�1 and �v�3 integrins and degradeOPG through CD138-mediated internalization. These cell-to-cell interactions greatly upregulate the production of a number of osteoclastogenic factors bystromal cells and early osteoblasts. RANKL enhances the differentiation of marrow macrophages to osteoclasts that undergo functional polarization in relationto the expression of a specific enzyme TRAcP and F-actin rearrangement in the cytoskeleton to form the sealing border of the membrane needed for adhesion tothe bone surface. Therefore, osteoclasts act as suckers and release a number of different enzymes to digest both organic and inorganic bone components. Basedon the chronic deregulation of osteoblasts in multiple myeloma, the excess of RANKL released within the marrow is considered the primary event driving theosteoclast hyperactivity that leads to uncontrolled bone resorption in MBD.

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2. Clinical monitoring of MBD

32 F. Silvestris et al. / Leukem

major transcriptional defect. However, soluble OPG is alsoeutralized by myeloma cells within the marrow through theirverexpression of syndecan-1 (CD138) molecules. Thesenclude a transmembrane proteoglycan enriched with hep-rin sulphate groups that binds OPG through its heparinomain, leading to its internalization and subsequent degra-ation within the lysosomal compartment of the malignantlasma cells [6]. Therefore, the transcriptional and post-ranslational decrease of OPG strongly reduces the potentialf neutralizing RANKL, and this results in the persistence ofredominant osteoclastogenic differentiation and hyperacti-ation.

Comparison of patients with active MBD and multiplesteolytic lesions with those with minimal skeleton involve-ent and inactive disease reveals an inverse correlation

etween marrow or serum levels of OPG with the severityf MBD [7,8]. Such a reverse RANKL/OPG ratio in MMndicates that OC hyperactivation is a major pathogenetic

echanism of MBD, though its primary event includes theeregulated control of pre-OBs and stromal cells in the pro-uction of osteoclastogenic factors.

Recent studies [9] have shown that RANKL is expressednd released by malignant plasma cells, which in turnncrease its marrow levels and accelerate OC differentiationnd maturation [10]. However, RANKL expression has noteen detected at protein and RNA levels either on primaryalignant plasma cells or cell lines by other investigators,ho suggest that RANKLs direct osteoclastogenic effect isrimarily exerted by cells of the OB lineage [11]. On thether hand, the amount of RANKL produced in vitro byyeloma cells is too low for an efficient reinforcement ofCs formation and activation, and may simply prevent their

poptosis.

.2. Defective OB function

The functional activity of stromal cells and early OBsithin the resorbed sites is permanently damaged followingerturbation by malignant plasma cells in the transcription ofheir own cell products. That OB function is severely impairedn MM patients is illustrated by the extreme paucity of osteo-alcin and bone morphogenetic proteins (BMPs), as productsf new bone formation in the erosive lacunae and osteolyticesions. However, the inability of OBs to repair the erosions isot only the effect of the altered cytokine homeostasis in thearrow, but also the result of multiple impaired mechanisms

irectly induced by the malignant plasma cells.We have recently described one of these mechanisms

12]. Chronic exposition to increased levels of inflamma-ory cytokines such as TNF-� and IFN-� in the marrowenders early OBs highly susceptible to apoptosis as theesult of upregulation of apoptogen receptors, including p55

nd p75 TNFRs, Fas and the DR4/DR5 complex as TRAILo-receptor. Apoptosis of early or inflamed OBs is directlyriggered by myeloma cells through presentation of eitheras-L or TRAIL. By preparing autologous co-cultures of OBs

Mre

earch 31 (2007) 129–138

ith autologous primary myeloma cells from patients withevere bone involvement, we demonstrated that the malig-ant plasma cells are powerfully toxic against OBs throughheir overexpression of apoptogen ligands. This mechanismas also corroborated in vivo by immunohistochemistry inone biopsies showing TUNEL-positive OBs juxtaposed toyeloma cells (Fig. 2).Myeloma cells also activate other mechanisms to sup-

ress OBs within the marrow. A major functional inhibitions induced by interfering with the Wnt-signaling pathway oftromal cells during their maturation to OBs. As depicted inig. 3, this pathway includes inhibition by Dkk1 (dickkopf1)rotein released by myeloma cells of lipoprotein receptor-elated protein (LRP) 5 and/or LRP6 receptors on the surfacef stromal cells [13]. This inhibiting factor, whose gene isverexpressed in patients with disseminated skeletal lesionsn parallel with increased circulating levels of the protein,nables both LPR5 and LPR6 receptors to transduce signalshrough the Wnt pathway, thus preventing the activation of theelative growth factors. The biochemical events induced byhis pathway include stabilization of cytoplasmic �-cateninith subsequent OB maturation. Impaired OB function is also

ttributable to direct inhibition of the Runx2/Cbfa1 transcrip-ion factor expressed by stromal cells [14]. This is revealedn vitro by the defective expression of osteocalcin, alkalinehosphatase, and collagen I mRNA and protein in humanre-OB cells co-cultured cell-to-cell with myeloma cells.unx2/Cbfa1 is essential for the maturation of stromal cells.

ts suppression in vivo is evident, since the bone biopsies ofatients with overt MBD disclose a dramatic paucity of stro-al cells expressing the transcription factor, either as protein

r mRNA [15].Other potential inhibitors include IL-11, which is probably

ecreted by the OBs themselves and is increased in a subset ofatients with severe skeletal disease, the IGF-binding proteinBP) 4 and the soluble Frizzled related protein (sFRP)-2 [16].GF-BP4 is produced by myeloma cells and inhibits the IGF-timulated growth of OBs, whereas sFRP-2 and -3, which alsoct as Wnt-signaling pathway inhibitors and are released byyeloma cells, block OB differentiation in vitro.Once deregulated by these inhibitory factors (partly listed

n Table 2), the OB function remains permanently ineffec-ive. Bone erosion sites, in fact, are rarely repaired even ifsteolysis is apparently halted in patients with prolongedemission after chemotherapy or combined treatments usingew biological drugs such as proteasome inhibitors, mono-lonal antibodies or anti-angiogenic molecules.

A striking imbalance of bone physiology is typical ofBD. It is usually downregulated by chemotherapy in

esponding patients, but requires constant monitoring tonsure early detection of its progression.

F. Silvestris et al. / Leukemia Research 31 (2007) 129–138 133

Fig. 2. Increased osteoblast apoptosis in MBD. Bone biopsy of a myeloma patient with active MBD showing apoptotic osteoblasts as fused cells facing plasmac ir positii

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ells within the spongy bone. These cells show dark nuclei in relation to then apoptotic cells.

.1. Diagnostic imaging

Radiography detects osteolytic lesions and illustrates theirrogression, but cannot provide a dynamic account of ongo-ng bone resorption and defective remodeling. Osteolysis

ay occur in most sites. It results in poorly marginated lacu-ae with no reactive bone, loss of medullary trabeculation

nd extensive endosteal erosion. Skeletal involvement maylso present as severe osteopenia and diffuse osteoporosis.his pattern of MBD is usually early and conventional radio-raphy may only detect a systemic loss of mineral content

Csos

ve reaction to the TUNEL method which reveals the fragmented chromatin

rimarily involving the axial skeleton. Finally, X-rays in theollow-up of MBD reveal both the enlargement of existingnd the appearance of new osteolytic lesions rather than pro-ression of osteopenia. Cortical destruction is also illustratedy CT imaging of contiguous tomographic planes.

99mTc bone scintigraphy is useful for monitoring the pro-ression of MBD. It is more sensitive than radiography and

T and allows total body assessment of dynamic erosion

ites. Even so, its specificity for MBD is poor. Magnetic res-nance imaging (MRI), on the other hand, is both highlyensitive and highly specific. It provides superior anatomical

134 F. Silvestris et al. / Leukemia Research 31 (2007) 129–138

Fig. 3. Molecular mechanisms of defective osteoblastogenesis in MBD. Myeloma cells release a number of inhibitory factors for osteoblast functions. Dkk1i the strb r transcf

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nterferes with the Wnt pathway by LPR membrane receptors and preventsone remodeling cells. Other inhibitors deregulate in stromal cells the majounctions leading to the maturation of stromal cells to osteoblasts.

etails in both soft tissue and bone and is thus indispensableor assessment of medullary canal invasion due to localiza-ion of myeloma in the vertebrae.

Positron emission tomography is currently undervaluation to determine its sensitivity in scanning the pro-ression of minimal bone erosions undetectable with otherethods.

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able 2everal inhibitory factors of osteoblastogenesis in myeloma bone disease

Cellular origin

ytokinesIFN-� Myeloma cellsTNF-� Stromal and myeloma ceTNF-� Stromal and myeloma ceIL-3 Marrow T cellsIL-11 Stromal and myeloma ce

poptogen ligandsFas-L Myeloma cellsTRAIL Myeloma cells

ther factorsDkkl Myeloma cellsIGF-BP4 Myeloma cellssFRP-2 and-3 Myeloma cellsInhibitors of Runx2/Cbfal Myeloma cells

IFN) interferon; (TNF) tumor necrosis factor; (IL) interleukin; (TRAIL) TNF-relateactor; (sFRP) soluble Frizzled related protein; (OB) osteoblasts.

uctural assemblage of �-catenin necessary for mature osteoblasts to act asription factor Runx-2/Cbfa1, which is essential to activate several cellular

.2. Clinical biochemistry

Fuller understanding of bone pathophysiology in MM has

hown that a number of biochemical markers specificallyeflect the extent of bone resorption or defective formation.

Both cellular and extracellular components of the skele-al matrix, including the collagen degradation products from

Biological effect

OB apoptosis upregulationlls OB apoptosis upregulationlls OB apoptosis upregulation

Inhibition of stromal cell maturationlls Inhibition of OB differentiation

OB cytotoxicityOB apoptosis upregulation

Inhibition of Wnt pathway of OB growthSuppression of IGF-mediated OB growthInhibition of Wnt-signaling pathwaySuppression of stromal cell maturation

d apoptosis inducing ligand; (Dkkl) dickkopfl protein; (IGF) insulin growth

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C activity, can be measured in MM. They include urinaryydroxyproline, hydroxylysine glycosides and cross-linkedmino-(NTX) and carboxy-(CTX) terminal telopeptides inerum. These amino acids are predominant in almost allenetic types of collagen. They are primarily metabolizedn the liver and excreted in the urine. Both NTX and CTXre released in sera as small type I collagen fragments thatass through the glomeruli into urine in relation to their smallize. NTX is specific for bone tissue breakdown and a directroduct of OC proteolysis. It is usually evaluated by meansf ELISAs employing antibodies to different epitopes of itslpha chain fragments. Similar assays are applied to measureoth serum and urine levels of CTX, whose increased excre-ion together with NTX reflects enhanced bone resorption inctive MBD.

Increasing interest has been devoted over the past fewears to serum levels of the tartrate resistant acid phos-hatase isoform type 5b (TRACP-5b) since they are relatedo the extent of OC activation in MBD. Both TRACP-5and -5b are produced and released in sera as macrophage-r OC-derived isoforms. TRACP-5b molecules are spreads complexes including �2-microglobulin and calcium, andirculate as catalytically active fragments that are progres-ively degraded in serum. Therefore, all active fragments inhe serum are freshly released by OCs and reflect the extentf concurrent resorption [17].

Cellular derivatives of OC activity in MBD have beenecently measured by several investigators. Attention haseen primarily directed to the circulating levels of MIP-� and RANKL. Serum MIP-1� is mainly secreted byoth malignant plasma cells and OBs [18,19]. It acts as aotent activator of OCs and induces their adhesion to maturesteocytes, as well as the formation, migration and differ-ntiation of OC progenitors in combination with RANKL.ncreased serum levels in newly diagnosed patients areirectly correlated with the number of osteolytic lesions and2-microglobulin levels. The poor prognosis of these patientstems from enhanced replication of their malignant plasmaells as the prime producers of MIP-1�, whereas its levelsre significantly lower in patients in remission and those withtable disease.

Circulating soluble(s) RANKL levels also reflect the activ-ty of MBD. The sRANKL/OPG ratio and the radiographicxtent of bone disease correlate with more than three oste-lytic lesions in different bones and in parallel with TRACP-b serum elevations [20]. In addition, OPG is almost unde-ectable in both marrow plasma and peripheral blood ofatients with newly diagnosed MM and disseminated oste-lytic lesions. By contrast, it is readily measured, though at aower magnitude than in healthy individuals, in patients withtable remission or inactive MBD.

Measurement of osteocalcin, a non-collagenous protein

ainly produced by OBs and activated chondrocytes, has

een recently suggested for bone remodeling evaluation.steocalcin is a 49-amino-acid protein with a MW of 11 kDa.

t contains a 23-residue hydrophobic signal peptide and

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earch 31 (2007) 129–138 135

26-residue pro-peptide. After cleavage of the hydropho-ic regions by a signal peptidase, the pro-osteocalcin is �-arboxylated, with subsequent removal of the pro-peptideefore the mature protein is secreted. A fraction of the newlyynthesized osteocalcin is released into the bloodstream byctivated OBs and is incorporated into the calcium bindingone matrix. Its serum levels are thus highest during OBepair after traumatic fractures or orthopedic surgery and aresually low in MM patients with active skeleton derange-ents.We have recently investigated several markers of bone

athophysiology to determine their potential application inhe evaluation of both resorbing and remodeling functionss compared to the activity state of MBD. Both osteocalcinnd OPG were selected to detect the OB activity, whereasIP-1� and RANKL levels were assumed to reflect the OC

unction. Data from these patients are presented in Fig. 4.atients with active MBD, whether untreated (group I) or

reated with conventional anti-MM chemotherapy and dis-laying one (group II) or several (group III) osteolytic lesions,xpressed higher median MIP-1� and RANKL levels thanhose in chemotherapy-induced remission with inactive boneisease (group IV), who indeed displayed a moderate increasef serum osteocalcin and higher OPG levels than the otherroups. These results are in agreement with those of othertudies [21] and support the clinical use of these markers inaking therapeutic decisions. For instance, patients without-ray-detectable MBD, but with increased serum levels ofIP-1� and RANKL, would benefit from bisphosphonates

n combination with first-line chemotherapy, whereas duringemission with normalized levels of OC activity markers andncreased serum OPG, their periodic administration could beelayed.

. Current treatments

Clinical studies are illustrating the efficacy of bispho-phonates (BPs) in inducing both symptomatic relief andetardation of MBD progression through their modulation ofyperactive osteoclastogenesis and reduction of its skeletalomplications [22,23]. These compounds display specificvidity for the metabolic sites of the mineral bone, where theynhibit dissolution of the hydroxyapatite crystals and down-egulate the major OC functions. The nitrogen (N)-containingPs, namely alendronate, ibandronate, pamidronate, rise-ronate and zoledronic acid, are more effective in reducingoth OC activity and survival through different mechanisms.fter their internalization, N-BPs interfere with the biosyn-

hetic mevalonate pathway by inhibiting a key enzyme,amely farnesyl diphosphonate (FPP) synthase. This resultsn deregulation of several intermediates enrolled in the

renylation of intracellular proteins active within the cellycle such as Ras, Rho and Rac [24], which are involved inhe regulation of cell proliferation, survival and cytoskeletalrganization. Inhibition of Ras signaling within OCs leads to

136 F. Silvestris et al. / Leukemia Research 31 (2007) 129–138

Fig. 4. Serum levels of bone metabolism markers in myeloma patients. Thirty-six patients were divided in four groups, namely newly diagnosed patients withactive MBD before any treatment (group I), with a single (group II) or multiple (group III) osteolytic lesions under treatment and in clinical remission (groupI activityM , the inca alues ar

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V) induced by conventional chemotherapy. A general defect of osteoblastIP-1� and RANKL were evident in patients with active MBD. By contrast

n apparent restoration of this cell lineage during the disease-free period. V

efects in their intracellular vesicle transport and their inabil-

ty to form the ruffled borders of their membrane needed toctivate bone resorption. N-BPs also act as potent inducersf apoptosis in OCs by producing an intracellular ATPetabolite that activates its mitochondrial pathway (Fig. 5).

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ig. 5. Mechanism of action of nitrogen-containing bisphosphonates (N-BPs). Thnhibit the farnesyl pyrophosphate enzyme. The inhibitory effect leads to defectiveo form the sealing zone in polarized osteoclasts. In addition, once deregulated, thsteoclast apoptosis.

as osteocalcin and OPG serum levels and, conversely, an increase of bothreased levels of osteoblast activity in patients in clinical remission reflectede compared to normal donor (ND) levels.

N-BPs exert a direct antitumor activity both in vitro and in

olid tumors and mouse MBD. Their mechanisms of actionnclude induction of apoptosis mediated by the caspase 3athway, and inhibition of several functions related to tumorrogression, such as neo-angiogenesis, cell adhesion and

ese compounds interfere with the mevalonate pathway in osteoclasts andprenylation of Ras, a cell cycle protein critical for rearranging the F-actine enzyme activates the production of a cytotoxic ATP analog that primes

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etachment of malignant cells from stroma following neutral-zation of their metalloproteases (MMP-2 and -3). A strikingeduction of osteolytic lesions and skeletal tumor burden haseen achieved in several mouse myelomas by repeated treat-ent with N-BPs.Injection of mice with 120 mg/kg of zoledronic acid sub-

utaneously twice weekly resulted in stabilization of ero-ions, retardation of new osteolytic lesions and reduction ofhe skeletal tumor burden as evidenced by serum paraproteinevels [25].

In a recent 2-year trial, the efficacy of zoledronic acid wasompared to that of pamidronate in a large number of patientsith MBD or with bone metastases from breast cancer aseasured by the occurrence of skeletal-related events (SRE)

uch as pathological fractures, requirement of radiotherapyr bone surgery [26]. The study demonstrated the superior-ty of zoledronic acid, which produced a 3-months delay inhe occurrence of SRE with comparable adverse effects andidney tolerability.

Studies on the molecular mechanisms involved in theathogenesis of MBD have also provided new therapeuticpproaches. Based on the major osteoclastogenic role exertedy RANKL, the Fc-RANK recombinant (r) construct haseen successfully used in animal models of bone metastasesrom solid tumors as well as in mouse myeloma models toaturate the excess of RANKL [27]. In addition, preliminarytudies in a small group of patients with MBD showed thebility of the rOPG product to reduce the urinary excretionf NTX and stabilize bone lesions over a period of severalonths [28]. Similar results in patients with bone metastases

rom breast cancer suggest that rOPG could be used in pre-entive treatment of bone erosive tumors by acting throughhe RANKL pathway.

Lastly, stem cell technology may be applicable to the treat-ent of MBD [29] since the ability of mesenchymal/stromal

tem cells (MSC) from bone marrow to differentiate in vitronto the osteogenic lineage in presence of conditioning mediaas been demonstrated [30]. Ex vivo MSC transfected withenes related to BMP-2, -4, and -7 produce stable transfec-omas in vitro that may induce heterotopic bone regenerationn animal models of skeleton diseases. Great interest is beingurrently devoted to the study of the mouse models of MBDuch as those of the 5T2 strain, to explore the ability of exivo BMP-transfected MSCs to regenerate new bone in ero-ive sites.

cknowledgments

This work was supported by FIRB 2001 and COFIN 2005rants from the Italian Ministry for Education, the Uni-ersities and Scientific Research (MIUR), Rome; a grant

rom the Associazione Italiana per la Ricerca sul CancroAIRC), Milan, and local funds from the University of Bari60%). Authors had no conflicts of interest in presenting datancluded in this work.

[

earch 31 (2007) 129–138 137

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