**3. Normal bone remodelling**

**2. Pathogenesis of multiple myeloma bone disease**

ting factor for the development of osteolytic lesions (see figure 1).

The reason for the excessive loss of bone mass observed in MM is multi factorial. For many years attention was primarily focused on the increase in bone degradation which is observed

Over the last decade however, it has become increasingly evident that impaired bone formation also plays an important role in MM bone disease. In monoclonal gammopathy of unknown significance (MGUS) and early stage MM with preserved bone structure, normal or even increased bone formation may be observed. With disease progression and development of osteolytic lesions bone formation becomes impaired, and this may be an important contribu‐

The interaction between the bone marrow microenvironment and the myeloma cells is also considered to be crucial. A large number of cytokines and chemokines, that regulate the activity of bone resorbing osteoclasts and bone forming osteoblasts, have been identified and studied in MM. Recently, a structure consisting of a flat layer of osteoblast lineage cells, that separates the bone surface from the bone marrow during bone remodelling, has been described. Disruption of this cell layer, called the bone remodelling compartment (BRC) canopy, allows direct contact betweenmyeloma cells andthe activebone remodellingcells, andthismayaffectbothcelltypes. OsteocyteshavebeensparselyinvestigatedinMM.However, a recent article illustrates that also

this type of cell may be important for a better understanding of MM bone disease [6].

**Bone resorption markers Bone formation markers**

a description of the bone remodeling compartment. University of Southern Denmark, 2008.

creased levels observed in early stages of MM.

With permission from the author; Søndergaard T. The effect of simvastatin on bone markers in multiple myeloma and

**Figure 1.** Number of studies evaluating biochemical markers of bone turnover in MM patients in a ten year period. Bone resorption markers are uniformly elevated, while the bone formation markers are more divergent, with in‐

**2.1. Introduction**

in the majority of MM patients.

218 Multiple Myeloma - A Quick Reflection on the Fast Progress

Osteoclasts are the cells responsible for bone resorption. They originate from the monocytemacrophage cell line. Differentiation of hematopoietic precursor cells into mature osteoclasts requires different environmental factors of which macrophage-colony stimulating factor (M-CSF) and receptor activator for NF-κB ligand (RANKL) play an essential role. The early step in osteoclastogenesis seems to be influenced by M-CSF [7], whereas RANKL initiates differ‐ entiation, cell fusion, and activation of mature osteoclasts [8]. During osteoclast development the cell replaces the nonspecific esterase activity with tartrate-resistant acid phosphatase isotype 5b (TRACP 5b), which is believed to be specific for osteoclasts. Osteoclastogenesis results in the formation of large multinucleated cells located on the bone surface where bone degradation takes place. Bone degradation is achieved by an active secretion of protons from the osteoclasts into the resorption pits. The protons decrease the pH and cause decalcification of the bone matrix [9]. After decalcification the collagen fibres are degraded mainly by the proteolytic enzymes cathepsin K and various matrix metalloproteinases [10].

Osteoblasts are responsible for the formation of new bone following osteoclast-mediated bone resorption. Osteoblasts originate from differentiated mesenchymal stem cells under the influence of Runt-related transcription factor (Runx2) and the wingless type signalling (Wnt) factors. Runx2 is required for the differentiation of mesenchymal cells into osteoblasts [11]. The Wnt-pathway mediates the formation of a complex, which in turn inhibits the proteasomal degradation of β-catenin. The increasing level of β-catenin has a stimulating effect on osteoblast differentiation and maturation [12]. The Wnt-pathway can be inhibited by Dickkopf 1 (DKK1), resulting in decreased bone formation.

Mature osteoblasts are lined in groups located along the newly resorbed bone. Placed on the resorption site, the osteoblasts secrete the components needed to generate bone matrix, mainly collagen type 1 [13]. The bone formation ends with calcification of the newly synthesized bone. During bone formation some osteoblasts are incorporated into the bone matrix and become osteocytes. Bone lining cells and the canopy cells are also of osteoblast lineage.

Activation of bone remodelling is not yet clearly understood. However, it is thought that osteocytes may, at least partly, be of importance for the activation of bone remodelling. Osteocytes in the bone matrix may respond to mechanical stimulation and via communication through their networks of canaliculli initiate bone resorption. Osteocyte death probably also plays a role in the recruitment of osteoclasts.

Bone remodelling takes place on bone surface where the osteoclasts and osteoblasts are covered by a canopy of flattened cells of osteoblast lineage [14;15]. The space between the canopy and the bone surface undergoing remodelling is named the bone remodelling com‐ partment (BRC). Disruption of the BRC canopy may impair bone remodelling [16]. Several factors of importance for the regulation of bone remodelling have been identified during the last decades. Within this chapter, we will only review some of the most important. The RANKL, RANK, and the decoy receptor osteoprotegerin (OPG) are probably the most significant factors in the regulation of normal physiological bone remodelling. RANK is expressed on the surface of osteoclast precursor cells, and as mentioned above, stimulation with RANKL is essential for osteoclastogenesis [17]. RANKL is expressed by osteoblasts and bone marrow stromal cells. OPG has a high affinity for RANKL and functions as the physiological inhibitor of RANKL [18]. Since osteoblasts can stimulate osteoclast activity through the expression of RANKL and inhibit it through the secretion of OPG, osteoblasts hold a key position in the coupling between bone formation and bone degradation. Another interesting regulator of bone degradation is macrophage inflammatory protein 1-α (MIP-1α). MIP-1α has been shown to be a potent activator of osteoclasts [19]. MIP-1α stimulates the activity and formation of osteoclasts indirectly by increasing the stromal cell expression of RANKL on the one hand [20; 21], but it also stimulates osteoclast formation independently of the RANKL system, though binding to the CCR1 or the CCR5 osteoclast receptor [21].

level of OPG, thus resulting in increased bone resorption [25]. The increased soluble RANKL/ OPG ratio has been shown to correlate with the extent of bone disease and even with overall survival [25;26]. In addition, myeloma cells stimulate bone degradation by the secretion of MIP-1α. In approximately 70% of MM patients, bone marrow serum levels of MIP-1α are elevated [27] and peripheral blood levels of MIP-1α have been found to correlate with bone

Bone Disease in Multiple Myeloma http://dx.doi.org/10.5772/55190 221

Vascular endothelial growth factor (VEGF) is known to be important for neovascularisation, but it probably also plays a role in the activation of osteoclasts in MM. VEGF has been demonstrated, in vitro, to act like macrophage colony-stimulating factor (M-CSF), thus inducing osteoclast differentiation [30]. Furthermore, a simultaneous blockade of VEGF and osteopontin has been shown to inhibit angiogenesis and bone resorption in co-cultures of myeloma cells and osteoclasts [31]. Taken together, these results indicate that VEGF could be of importance in bone resorption, and since the majority of myeloma cells can secrete VEGF it has been suggested that VEGF may support osteoclastic bone resorption in MM [32]. Inter‐ leukin-6 (IL-6), stromal-derived factor-1α, tumor necrosis factor-α, and interleukin-11 are other examples of cytokines known to stimulate osteoclasts, which are suggested to be of importance

The myeloma cells do not only affect the osteoclasts indirectly through the secretion of cytokines into the bone marrow microenvironment, but a direct contact between myeloma cells and bone marrow stromal cells or osteoclasts also seems to be an important factor in the

Disruption of the BRC canopy is a frequent finding in MM. This breakdown of the BRC canopy allows a direct contact between the myeloma cells and the osteoclasts and osteoblasts involved in bone remodelling. This event probably contributes to impaired bone formation and enhanced bone resorption [16]. The extent of BRC canopy disruption in a histomorphometric study of iliac crest biopsies was found to correlate with the magnitude of osteolytic lesions in patients with MM [16]. Direct contact between human myeloma cells and bone marrow stromal cells or pre-osteoblasts tested in a co-culture system resulted in a marked decrease in the production of OPG, and thereby an imbalance in the RANKL/OPG ratio resulting in increased bone degradation [35]. Cell to cell contact between myeloma cells and bone marrow stromal cells has also been demonstrated to induce the secretion of IL-6 by bone marrow stromal cells [31]. IL- 6 stimulates osteoclast formation and also has a promoting effect on myeloma cell proliferation [36]. It has also been suggested that myeloma cells can fuse with osteoclasts to create myeloma-osteoclast hybrid cells that may more aggressively erode bone than non-

Co-cultures of myeloma cells and osteoclasts have demonstrated an increased viability of the myeloma cells caused by the direct cell to cell contact with osteoclasts [38]. Osteoclasts also produce factors capable of promoting myeloma cell growth, including IL-6 [39] and insulinlike-growth factor-1 (IGF-1) [40]. Osteoclasts can also support myeloma cell growth through the production of angiogenic factors, and the direct contact between myeloma cells and osteoclasts in co-cultures has been shown to enhance vascular tubule formation [41]. In animal models the inhibition of osteoclast activity with recombinant OPG or bisphosphonates has

disease and overall survival [28;29].

in the development of MM bone disease [33;34].

development of MM bone disease.

hybrid osteoclasts [16;37].

**Figure 2.** Normal bone remodelling and bone remodelling in multiple myeloma. A: Osteocytes sense mechanical stress and activate bone remodelling. B: Osteoclast precursors differentiate into mature multinucleated osteoclasts. C: The osteoclasts resorb bone matrix. D: Following bone resorption mononucleated osteoblasts lay down new bone in the resorbed area. E: During bone formation of new bone, osteoblasts are imbedded in the new bone matrix and dif‐ ferentiate into osteocytes. F: The bone remodelling takes place beneath a canopy of cells belonging to the osteoblast lineage G: Malignant plasma cells disrupt the bone remodelling compartment canopy and H: Increase osteoclastogen‐ esis and I: Decrease osteoblastogenesis.

#### **4. Abnormal bone remodelling in multiple myeloma**

Increased bone degradation is an early event in MM. Retrospective studies using bone histomorphometry on bone marrow biopsies from patients diagnosed with MGUS harvested three to twelve months before these patients developed MM, were found to have increased bone degradation compared with MGUS patients who did not progress to MM during the first year after MGUS was diagnosed [22]. In MM both the number and the activity of the osteoclasts are found to be increased, and this may result in either focal or more diffuse loss of bone matrix when not compensated for by an equal increase in bone formation [23;24].

Several factors of importance for the development of MM bone disease have been identified during the last decades. The RANK/RANKL/OPG system is one of the most significant. In normal bone remodelling the RANKL/OPG ratio is tightly balanced. In MM the RANKL/OPG ratio is increased, both due to an elevated level of RANKL and as a result of a decrease in the level of OPG, thus resulting in increased bone resorption [25]. The increased soluble RANKL/ OPG ratio has been shown to correlate with the extent of bone disease and even with overall survival [25;26]. In addition, myeloma cells stimulate bone degradation by the secretion of MIP-1α. In approximately 70% of MM patients, bone marrow serum levels of MIP-1α are elevated [27] and peripheral blood levels of MIP-1α have been found to correlate with bone disease and overall survival [28;29].

of osteoclast precursor cells, and as mentioned above, stimulation with RANKL is essential for osteoclastogenesis [17]. RANKL is expressed by osteoblasts and bone marrow stromal cells. OPG has a high affinity for RANKL and functions as the physiological inhibitor of RANKL [18]. Since osteoblasts can stimulate osteoclast activity through the expression of RANKL and inhibit it through the secretion of OPG, osteoblasts hold a key position in the coupling between bone formation and bone degradation. Another interesting regulator of bone degradation is macrophage inflammatory protein 1-α (MIP-1α). MIP-1α has been shown to be a potent activator of osteoclasts [19]. MIP-1α stimulates the activity and formation of osteoclasts indirectly by increasing the stromal cell expression of RANKL on the one hand [20; 21], but it also stimulates osteoclast formation independently of the RANKL system, though binding to

**Figure 2.** Normal bone remodelling and bone remodelling in multiple myeloma. A: Osteocytes sense mechanical stress and activate bone remodelling. B: Osteoclast precursors differentiate into mature multinucleated osteoclasts. C: The osteoclasts resorb bone matrix. D: Following bone resorption mononucleated osteoblasts lay down new bone in the resorbed area. E: During bone formation of new bone, osteoblasts are imbedded in the new bone matrix and dif‐ ferentiate into osteocytes. F: The bone remodelling takes place beneath a canopy of cells belonging to the osteoblast lineage G: Malignant plasma cells disrupt the bone remodelling compartment canopy and H: Increase osteoclastogen‐

Increased bone degradation is an early event in MM. Retrospective studies using bone histomorphometry on bone marrow biopsies from patients diagnosed with MGUS harvested three to twelve months before these patients developed MM, were found to have increased bone degradation compared with MGUS patients who did not progress to MM during the first year after MGUS was diagnosed [22]. In MM both the number and the activity of the osteoclasts are found to be increased, and this may result in either focal or more diffuse loss of bone matrix

Several factors of importance for the development of MM bone disease have been identified during the last decades. The RANK/RANKL/OPG system is one of the most significant. In normal bone remodelling the RANKL/OPG ratio is tightly balanced. In MM the RANKL/OPG ratio is increased, both due to an elevated level of RANKL and as a result of a decrease in the

**4. Abnormal bone remodelling in multiple myeloma**

when not compensated for by an equal increase in bone formation [23;24].

the CCR1 or the CCR5 osteoclast receptor [21].

220 Multiple Myeloma - A Quick Reflection on the Fast Progress

esis and I: Decrease osteoblastogenesis.

Vascular endothelial growth factor (VEGF) is known to be important for neovascularisation, but it probably also plays a role in the activation of osteoclasts in MM. VEGF has been demonstrated, in vitro, to act like macrophage colony-stimulating factor (M-CSF), thus inducing osteoclast differentiation [30]. Furthermore, a simultaneous blockade of VEGF and osteopontin has been shown to inhibit angiogenesis and bone resorption in co-cultures of myeloma cells and osteoclasts [31]. Taken together, these results indicate that VEGF could be of importance in bone resorption, and since the majority of myeloma cells can secrete VEGF it has been suggested that VEGF may support osteoclastic bone resorption in MM [32]. Inter‐ leukin-6 (IL-6), stromal-derived factor-1α, tumor necrosis factor-α, and interleukin-11 are other examples of cytokines known to stimulate osteoclasts, which are suggested to be of importance in the development of MM bone disease [33;34].

The myeloma cells do not only affect the osteoclasts indirectly through the secretion of cytokines into the bone marrow microenvironment, but a direct contact between myeloma cells and bone marrow stromal cells or osteoclasts also seems to be an important factor in the development of MM bone disease.

Disruption of the BRC canopy is a frequent finding in MM. This breakdown of the BRC canopy allows a direct contact between the myeloma cells and the osteoclasts and osteoblasts involved in bone remodelling. This event probably contributes to impaired bone formation and enhanced bone resorption [16]. The extent of BRC canopy disruption in a histomorphometric study of iliac crest biopsies was found to correlate with the magnitude of osteolytic lesions in patients with MM [16]. Direct contact between human myeloma cells and bone marrow stromal cells or pre-osteoblasts tested in a co-culture system resulted in a marked decrease in the production of OPG, and thereby an imbalance in the RANKL/OPG ratio resulting in increased bone degradation [35]. Cell to cell contact between myeloma cells and bone marrow stromal cells has also been demonstrated to induce the secretion of IL-6 by bone marrow stromal cells [31]. IL- 6 stimulates osteoclast formation and also has a promoting effect on myeloma cell proliferation [36]. It has also been suggested that myeloma cells can fuse with osteoclasts to create myeloma-osteoclast hybrid cells that may more aggressively erode bone than nonhybrid osteoclasts [16;37].

Co-cultures of myeloma cells and osteoclasts have demonstrated an increased viability of the myeloma cells caused by the direct cell to cell contact with osteoclasts [38]. Osteoclasts also produce factors capable of promoting myeloma cell growth, including IL-6 [39] and insulinlike-growth factor-1 (IGF-1) [40]. Osteoclasts can also support myeloma cell growth through the production of angiogenic factors, and the direct contact between myeloma cells and osteoclasts in co-cultures has been shown to enhance vascular tubule formation [41]. In animal models the inhibition of osteoclast activity with recombinant OPG or bisphosphonates has resulted in an increased in survival of mice inoculated with myeloma cells [42;43] but the clinical data from myeloma patients treated with bisphosphonates have been less consistent [44-48]. Nevertheless, the existence of a vicious cycle of bone resorption and tumour growth in patients with MM seems plausible and may be supported by the demonstration of a survival advantage in patients treated with zoledronic acid in the MRC IX trial [49].

trabecular bone mass must be absent for a lesion to become detectable. Computed tomogra‐ phycanincreasethesensitivityatthecostofhigherradiationexposure.Bothmodalities,however, onlyprovide static informationconcerningthe accumulatedbonedisease.Biochemicalmarkers of bone turnover can provide dynamic information concerning the velocity of bone turn-over at any given time point, and can be measured from either blood or urine samples. Furthermore, bone formation and bone resorption can be evaluated separately. Bone markers can be divid‐ ed into two categories: they are either collagen fragments released during the formation or destruction of the collagen triple helix structure of which bone consists, or they are enzymes released form either osteoblasts or the osteoclasts (see figure 3). Bone resorption markers from thefirstgroupincludethecross-linkedtelopeptidesoftype-1collagenNTX,CTX,ICTPandDPD (Table1).Theyareproductsofosteoclast-mediateddegradationof collagenandthereforereflect bone resorption. Bone formation markers from this group include PINP and PINC (Table 1). These markers are products of the cleavage process of procollagen into collagen and therefore the measured levels will reflect the amount of newly formed bone matrix. The second group of bone markers include TRACP-5b, bALP and OC (Table 1). TRACP-5b is secreted by osteo‐ clasts and used as a marker of osteoclast number and activity, whereas bALP and osteocalcin are produced by osteoblasts and used as markers of osteoblast number and activity. The levels of bone markers have been shown to correlate with the degree of bone resorption or bone formation using classical bone histomorphometry [54;55]. Furthermore, bone resorption markers decrease when treatment with anti-resorptive drugs is initiated [56]. Conversely, the discontinuationofanti-resorptivedrugsleadstoariseinboneresorptionmarkers[57].However, when using biochemical markers it is important to be aware of the fact that the level of mark‐ ersmaybeinfluencedbyanumberoffactors, suchasage,gender,drugs,renal-andliverfunction ordiet.Especiallythecollagen-mediatedmarkersaresensitivetofoodintake.Despitetheinterest inbonemarkers,thereisstillnoconsensusonhowtheyshouldbeusedtomonitordiseaseactivity

**Bone marker Abbreviation Type Analytical specimen**

Deoxypyridinoline DPD Bone resorption marker Serum, Urine

Bone-specific alkaline phosphatase bALP Bone formation marker Serum Osteocalcin OC Bone formation marker Serum Procollagen type-1 N-propeptide PINP Bone formation marker Serum Procollagen type-1 C-propeptide PICP Bone formation marker Serum

CTX Bone resorption marker Serum, Urine

NTX Bone resorption marker Serum, Urine

ICTP Bone resorption marker Serum

osteoclast activity

Serum

Bone Disease in Multiple Myeloma http://dx.doi.org/10.5772/55190 223

TRACP-5b Bone resorption marker

and response to treatment in MM [58].

C-terminal cross-linking telopeptide

N-terminal cross-linking telopeptide

C-terminal cross-linking telopeptide of type-1 collagen generated by

Tartrate-resistant acid phosphatase

**Table 1.** Biochemical markers of bone turnover

of type-1 collagen

of type-1 collagen

metalloproteinase

isotype 5b

Bone disease in MM is not only caused by an increased bone resorption, but the formation of new bone may also be affected. A reduced recruitment of osteoblasts, as well as reduced mineral deposition has been observed using histological methods in patients with MM [22]. In early stage of MM the number and activity of the osteoblasts can be increased but a marked decrease occurs as the plasma cell infiltration progresses [50]. Disruption of the BRC canopy in MM may be an important cause of the uncoupling of bone resorption and bone formation, with the result that bone resorption is not followed by bone formation or that the bone formation process is delayed or abolished [16]. Human plasma cells purified from bone marrow biopsies of MM patients have been found to express the gene for DKK1, and immu‐ nohistochemical analysis of bone marrow biopsies have shown that myeloma cells contain DKK1 [51]. In addition, blood and bone marrow serum levels of DKK1 have been demonstrated to be elevated in patients with MM bone disease [51]. Since DKK1 is believed to inhibit the stimulation of osteoblastogenesis via the Wnt-pathway this might cause impaired bone formation. Runx2 may also be affected by myeloma cells. Runx2 is required for osteoblast differentiation. The expression of Runx2 by mesenchymal cells has been found to decrease after direct cell to cell contact with myeloma cells in co-cultures [52].

Osteocytes have not been widely investigated, and their involvement in MM bone disease is unknown. Histological examination of compact bone from MM patients shows a significant change in the morphology of osteocytes and their lacunae [53]. Likewise, a major change in the gene expression profile of osteocytes in MM has also been observed. This indicates that osteocytes are markedly affected in MM. A recently published study showed that MM patients had significantly smaller numbers of viable osteocytes compared to healthy individuals [6]. Likewise MM patients with bone lesions were found to have a smaller number of viable osteocytes compared with MM patients without bone lesions. The amount of viable osteocytes was found to be negatively correlated with the number of osteoclasts and the authors suggest an involvement of the osteocytes in MM-induced osteoclast formation [6].

Futhermore, healing of bone lesions in MM bone disease does not occur frequently, even in patients who respond well to anti-myeloma treatment. It remains unclear why bone remod‐ elling does not normalise when the influence from myeloma cells disappears after successful treatment. It may be due to irreversible damage of key elements in the bone formation process (i.e. the BRC).

## **5. Biochemical markers of bone turnover**

Conventional radiography has for many years been the standard method for the diagnosis of myeloma bone disease. This modality, however, suffers from a low sensitivity, since 30% of the

trabecular bone mass must be absent for a lesion to become detectable. Computed tomogra‐ phycanincreasethesensitivityatthecostofhigherradiationexposure.Bothmodalities,however, onlyprovide static informationconcerningthe accumulatedbonedisease.Biochemicalmarkers of bone turnover can provide dynamic information concerning the velocity of bone turn-over at any given time point, and can be measured from either blood or urine samples. Furthermore, bone formation and bone resorption can be evaluated separately. Bone markers can be divid‐ ed into two categories: they are either collagen fragments released during the formation or destruction of the collagen triple helix structure of which bone consists, or they are enzymes released form either osteoblasts or the osteoclasts (see figure 3). Bone resorption markers from thefirstgroupincludethecross-linkedtelopeptidesoftype-1collagenNTX,CTX,ICTPandDPD (Table1).Theyareproductsofosteoclast-mediateddegradationof collagenandthereforereflect bone resorption. Bone formation markers from this group include PINP and PINC (Table 1). These markers are products of the cleavage process of procollagen into collagen and therefore the measured levels will reflect the amount of newly formed bone matrix. The second group of bone markers include TRACP-5b, bALP and OC (Table 1). TRACP-5b is secreted by osteo‐ clasts and used as a marker of osteoclast number and activity, whereas bALP and osteocalcin are produced by osteoblasts and used as markers of osteoblast number and activity. The levels of bone markers have been shown to correlate with the degree of bone resorption or bone formation using classical bone histomorphometry [54;55]. Furthermore, bone resorption markers decrease when treatment with anti-resorptive drugs is initiated [56]. Conversely, the discontinuationofanti-resorptivedrugsleadstoariseinboneresorptionmarkers[57].However, when using biochemical markers it is important to be aware of the fact that the level of mark‐ ersmaybeinfluencedbyanumberoffactors, suchasage,gender,drugs,renal-andliverfunction ordiet.Especiallythecollagen-mediatedmarkersaresensitivetofoodintake.Despitetheinterest inbonemarkers,thereisstillnoconsensusonhowtheyshouldbeusedtomonitordiseaseactivity and response to treatment in MM [58].


**Table 1.** Biochemical markers of bone turnover

resulted in an increased in survival of mice inoculated with myeloma cells [42;43] but the clinical data from myeloma patients treated with bisphosphonates have been less consistent [44-48]. Nevertheless, the existence of a vicious cycle of bone resorption and tumour growth in patients with MM seems plausible and may be supported by the demonstration of a survival

Bone disease in MM is not only caused by an increased bone resorption, but the formation of new bone may also be affected. A reduced recruitment of osteoblasts, as well as reduced mineral deposition has been observed using histological methods in patients with MM [22]. In early stage of MM the number and activity of the osteoblasts can be increased but a marked decrease occurs as the plasma cell infiltration progresses [50]. Disruption of the BRC canopy in MM may be an important cause of the uncoupling of bone resorption and bone formation, with the result that bone resorption is not followed by bone formation or that the bone formation process is delayed or abolished [16]. Human plasma cells purified from bone marrow biopsies of MM patients have been found to express the gene for DKK1, and immu‐ nohistochemical analysis of bone marrow biopsies have shown that myeloma cells contain DKK1 [51]. In addition, blood and bone marrow serum levels of DKK1 have been demonstrated to be elevated in patients with MM bone disease [51]. Since DKK1 is believed to inhibit the stimulation of osteoblastogenesis via the Wnt-pathway this might cause impaired bone formation. Runx2 may also be affected by myeloma cells. Runx2 is required for osteoblast differentiation. The expression of Runx2 by mesenchymal cells has been found to decrease

Osteocytes have not been widely investigated, and their involvement in MM bone disease is unknown. Histological examination of compact bone from MM patients shows a significant change in the morphology of osteocytes and their lacunae [53]. Likewise, a major change in the gene expression profile of osteocytes in MM has also been observed. This indicates that osteocytes are markedly affected in MM. A recently published study showed that MM patients had significantly smaller numbers of viable osteocytes compared to healthy individuals [6]. Likewise MM patients with bone lesions were found to have a smaller number of viable osteocytes compared with MM patients without bone lesions. The amount of viable osteocytes was found to be negatively correlated with the number of osteoclasts and the authors suggest

Futhermore, healing of bone lesions in MM bone disease does not occur frequently, even in patients who respond well to anti-myeloma treatment. It remains unclear why bone remod‐ elling does not normalise when the influence from myeloma cells disappears after successful treatment. It may be due to irreversible damage of key elements in the bone formation process

Conventional radiography has for many years been the standard method for the diagnosis of myeloma bone disease. This modality, however, suffers from a low sensitivity, since 30% of the

advantage in patients treated with zoledronic acid in the MRC IX trial [49].

222 Multiple Myeloma - A Quick Reflection on the Fast Progress

after direct cell to cell contact with myeloma cells in co-cultures [52].

an involvement of the osteocytes in MM-induced osteoclast formation [6].

**5. Biochemical markers of bone turnover**

(i.e. the BRC).

pathological fractures [63]. In 1996 and 1998, Berenson *et al.* published two studies, in which patients were randomised to placebo or the amino-bisphosphonate pamidronate. A significant effect was observed with regard to reduced pain, fewer skeletal related events, and improved quality of life [64;65]. Initially, no effect could be observed in overall survival, however using a Cox multivariable regression analysis a slight increase in overall survival was observed for a subgroup of patients. A subsequent phase III trial, comparing the more potent bisphonate zoledronic acid with pamidronate in breast cancer patients with bone metastases and MM patients, demonstrated a superiority of zoledronic acid over pamidronate in reducing skeletal events in the breast cancer group but not in the MM sub-population. No difference was observed in overall survival [66]. Later publications indicated that there could be an effect on overall survival but only with the most potent bisphosphonates [67-70]. In 2010 a large metaanalysis concluded that there was no effect on overall survival in MM provided by bisphosph‐ onates in general [71]. However, later the same year the large MRC IX trial, reported that zoledronic acid was superior to the non-nitrogen containing bisphosphonate clodronate, not only with regard to the control of bone disease, but zoledronic acid also increased overall survival by 5.5 months [49]. Because of the MRC IX data, an updated version of the metaanalysis was published in 2012. Still, no significant effect on overall survival was observed for bisphosphonates in general, but "meta regression analysis indicated that the beneficial effect of bisphosphonates on mortality in patients with MM may be a function of drug potency, with

Bone Disease in Multiple Myeloma http://dx.doi.org/10.5772/55190 225

Bisphosphonates are potential nephrotoxic compounds and dosage adjustment according to

In 2003, it was reported for the first time, that exposure to bisphosphonates could also cause osteonecrotic lesions, especially in the oral cavity. This complication was termed bisphosph‐ onate-associated osteonecrosis of the jaw (BON) [74]. BON is commonly observed after surgical dental procedures, e.g. tooth extractions, but spontaneous cases do occur [75]. The incidence of BON increases with treatment duration [76], as well as with the potency of the bisphosph‐ onate used [77]. The aetiology of BON remains controversial. One possible explanation could be that the profound suppression of osteoclast activity results in the accumulation of micro‐ fractures in the bone. This explanation is in accordance with the fact that BON incidence increases with treatment duration and potency of bisphosphonate type and that BON is also observed after treatment with denosumab, a monoclonal antibody that inhibits osteoclast activity by binding to RANKL. It has also been suggested that BON may occur because of the anti-angiogenic effects of bisphosphonates [78]. Indeed, BON seems to be more commonly observed in patients receiving other anti-angiogentic compounds such as thalidomide [77]. Thirdly, it has been speculated that the frequent findings of actinomycosis in the lesions may be part of the pathogenesis and not only a secondary event, especially since prophylactic antibiotics during dental procedures seem to reduce the incidence of BON [79]. Recently, osteomalacia, which in adults is often a consequence of vitamin D deficiency, has been suggested as a risk factor for BON [80]. Once established BON is difficult to cure, and surgical treatment may worsen the situation [75]. Case-reports suggest several treatment modalities, including low-level laser therapy [81;82], hyperbaric oxygen treatment [83], long-term

zoledronate being the most potent" [72].

creatinine clearance are required [73].

**Figure 3.** Biochemical markers of bone remodelling can be divided into markers reflecting bone resorption (left) and marker reflecting bone formation (right). They can also be divided in markers reflecting a change in the collagen ma‐ trix (upper part) or markers reflecting the activity of bone resorbing or bone forming cells (lower part).
