Myeloma Bone Lesions Research Articles

The EphrinB2/EphB4 Axis Is Dysregulated in Osteoprogenitors from Myeloma Patients and Its Activation by EphrinB2-Fc or EhpB4-Fc Affects Myeloma Bone Disease and Tumor Growth in Vivo

Induction of osteolytic bone lesions in myeloma (MM) is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Recent studies indicate that in addition to role in cell adhesion, repulsion and neovascularization, bidirectional signaling between the cell surface molecules EphrinB2 and EphB4 also mediates the coupling between osteoblasts and osteoblasts. While mesenchymal stem cells (MSCs) and osteoblasts express the ligand EphrinB2 land its receptor, EphB4, osteoclasts and their precursors mainly express EphrinB2. Forward signaling in MSCs promotes osteogenic differentiation and reverse signaling in osteoclast precursors inhibits their differentiation. The aims of the study were to investigate whether the EphrinB2/Eph4 axis is dysregulated in MM osteoprogenitors and whether activation of this axis in myelomatous bone by EphrinB2-Fc or EphB4-Fc affects MM bone disease, angiogenesis and tumor growth. MSCs were generated from bone marrow of healthy donors (n=5) and patients with MM (n=13). Gene expression was determined by qRT-PCR. MSCs from MM patients had reduced expression of EphrinB2 (EFNB2) by 61±6% (p<0.02) and EphB4 by 60±10% (p<0.02) than expression levels of these molecules in MSCs from healthy donors. Expression of other EFN and EPH B genes were detected and similarly expressed in patients and donors MSCs. Differentiation of MSCs from MM patients into osteoblasts resulted in upregulation of EFNB2 and downregulation of EPHB4. MM cell lines and primary MM plasma cells expressed low to undetectable levels of this family of genes. We exploited our SCID-hu system for primary MM to study the consequences of activation of forward signaling by EphrinB2-Fc or reverse signaling by EphB4-Fc on MM-induced bone disease and MM growth. Twelve SCID-hu mice were engrafted with MM cells from a patient with active MM. Upon detection of MM growth (by human Ig ELISA) and bone disease (radiographically), hosts were locally treated with Fc (control), EphrinB2-Fc or EPHB4 (4 mice/group) for 4 weeks using Alzet pump that continually released 0.11 μg/hour of each compound. While in Fc-treated hosts BMD of the implanted bone was reduced by 8±3% from pretreatment levels, it was increased by EphrinB2-Fc and EPhB4-Fc by 15±8% (p<0.03 vs. Fc) and 2±1% (p<0.02 vs. Fc) from pretreatment levels, respectively. At experiment's end levels of human Ig in mice sera were increased by 308±99% and 244±86% from pretreatment levels in Fc- and EphrinB2- Fc groups, respectively, while were reduced by 92±1% (p<0.02 vs. Fc) from pretreatment levels in EphB4-Fc group. In myelomatous bones, EphB4-Fc and EphrinB2-Fc increased the numbers of osteoblasts by >3 folds (p<0.004) while EphB4-Fc, but not EphrinB2-Fc, reduced osteoclast numbers by 5 folds (p<0.01 vs. Fc group). The numbers of CD34-reactive neovessels were reduced by 2 folds following treatment with EphB4-Fc (p<0.03) and were increased by 2.5 folds following treatment with EphrinB2-Fc (p<0.05). Our study suggests that downregulation of EphrinB2 and EhpB4 in MSCs from MM patients contributes to their impaired osteogenic differentiation and that treatment with EphrinB2-Fc or EphB4-Fc helps restore coupling of bone remodeling in myelomatous bones. The results also indicate that EphB4-Fc treatment is an effective approach to simultaneously inhibit MM and its associated bone disease.

Osteoclasts in Myeloma Are Derived from Gr-1+CD11b+ Myeloid Immune Suppressor Cells of the Bone Marrow Niche in Vivo

When cancer cells are resident in bone, they initiate a vicious cycle with osteoclasts (OCs) which perpetuates their growth and aggressive behavior. OCs are critical for the maintenance of the vicious cycle, since they control not only bone destruction associated with cancer, but also the aggressive behavior of tumor cells. It has recently been recognized that tumor cells grow in distant sites because they induce non-malignant cells to establish a “pre-metastatic niche” for tumor cells to later engraft. But nothing is yet known for bone. Primitive bone marrow progenitor cells, called myeloid immune suppressor cells (MISCs), which suppress immune reactivity, are important niche components. MISCs belong to the myelomonocytic lineage with surface markers of Gr-1 and CD11b. We hypothesize that MISCs are precursors of OCs recruited by tumors to assist in the establishment of the vicious cycle. To test this hypothesis, we used the well-characterized 5TGM1 murine myeloma model. 5TGM1-GFP tagged myeloma cells were inoculated via tail vein. The proportion of Gr-1+CD11b+ cells in bone marrow and spleen were assessed by FACS. On week 4 after tumor cell inoculation, %Gr-1+CD11b+ cells were significantly greater in tumor-bearing mice compared with controls (60.9±7.8% vs 37.7±8.6% p<0.05 in marrow; 21.1±4.84% vs 2.4±0.85% p<0.05 in spleen) and paralleled the myeloma burden in bone and spleen. We sorted the Gr-1+CD11b+ cells from the spleens by using magnetic microbeads. MISCs formed multinucleated TRAP positive OCs in medium containing M-CSF (25ng/ml) and RANKL (50ng/ml). The number of OCs derived from tumor MISCs was dramatically greater than those from control mice after 14 day culture (13.4±2.1 vs 1±0.7 per 100x field). MISCs expressed the αv chain of the vitronectin receptor (CD51) and the calcitonin receptor which are specific markers for OCs as they differentiated into multinucleated TRAP+ cells. Only MISCs from tumor-bearing mice and not MISCs from control mice caused resorption pits on dentine discs, demonstrating they were functional OCs. To study the in vivo differentiation of MISCs, we bred lacZ generalized C57 B6 mice with Rag2−/− immune compromised mice to generate lacZ+/− Rag2−/− mice. MISCs were sorted from lacZ+/− Rag2−/− myeloma mice. lacZ positive MISCs were co-injected with 5TGM1 cells to Rag2−/− mice. On day 10 after injection, lacZ+ multinucleated cells could be seen on endosteal surface beneath growth plate by X-gal staining. Those lacZ+ cells were also TRAP+, indicating they were OCs. Treatment of mice with zoledronic acid 100ug/kg s.c. 2/week for 4 weeks reduced % MISCs in tumor bearing mice and impaired the capacity of MISCs to form OCs in vitro (OC# 42.4±4.0 vs 25.6±3.5 per 100x field). Our data suggest that MISCs are increased significantly in marrow and spleen of myeloma-bearing mice and parallel the appearance of lytic bone lesions. These MISCs differentiate avidly and rapidly into functional OCs in vitro as well as in vivo. Zoledronic acid impairs both MISCs, the OC precursors, and mature OCs. These results have a number of implications, including the possibility of reducing bone lesions in myeloma and other malignancies by depleting specific subpopulations of osteoclast precursors.

Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

AbstractMultiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell–derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Link between osteoclastogenesis, angiogenesis and myeloma expansion

Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression. Myeloma cells and stromal cells secrete angiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Of note, osteoclasts constitutively secrete a large amount of a proangiogenic factor, osteopontin, which cooperates with VEGF from myeloma cells to enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. Therefore, a close link between myeloma cells, osteoclasts and vascular endothelial cells can be established in myeloma bone lesions, thereby forming a vicious cycle between bone destruction, angiogenesis and myeloma expansion.

多発性骨髄腫に対するビスフォスフォネート投与に関連したと考えられる上顎骨壊死の１例

Bisphosphonates (BPs) inhibit bone resorption and are widely used to treat skeletal complications in patients with the bone metastasis from malignant tumors, osteoporosis, and bone lesions of multiple myeloma. Osteonecrosis of the jaw (ONJ) has been associated with the use of BP. In Japan, zoledronic acid has been used to treat bone lesions of multiple myeloma and bone metastasis from malignant tumors since April 2006. Zoledronic acid has strong bone antiresorptive activity as compared with other BPs such as pamidronate. The number of patients given zoledronic acid and the frequency of ONJ are expected to increase in Japan.We report a patient with ONJ who was given zoledronic acid to prevent bone lesions of multiple myeloma. A 78-year-old man was referred to our hospital to undergo evaluation of bilateral asymptomatic bone exposure of the maxillary molar regions. MR imaging showed a low-signal-density area in the left maxillary molar region. Histopathologically, a biopsy specimen showed sequestration with attachment of bacteria. A final diagnosis of ONJ was made. Conservative follow-up is being continued, and no symptoms have developed.

Skeletal Imaging and Management of Bone Disease

Up to 90% of patients with multiple myeloma develop bone lesions. The lesions are purely osteolytic because of increased osteoclast activity and markedly suppressed or absent osteoblast activity. The "gold standard" for imaging myeloma bone lesions is the metastatic bone survey. However, plain radiographs are relatively insensitive and can only demonstrate lytic disease when 30% of trabecular bone loss has occurred. Technicium-99m bone scanning is not appropriate for evaluating myeloma patients since bone scans underestimate the extent of bone involvement in patients with myeloma. The limited reproducibility of bone surveys have led to the use of computerized tomography (CT) scanning, magnetic resonance imaging (MRI) and positron emission tomography (PET) scans to evaluate the extent of bone disease. CT scans are more sensitive than plain radiographs for detecting small lytic lesions, and MRI scans detect marrow involvement by the tumor. PET scans have been used to detect bone lesions in patients with myeloma, are more sensitive than plain radiographs, and have the same sensitivity as MRIs for detecting bone disease in the spine and pelvis. Treatment of myeloma bone disease involves treatment of the underlying malignancy and its manifestations. Current treatments that will be discussed include bisphosphonate therapy, kyphoplasty, vertebroplasty, radiation therapy, and novel agents to suppress osteoclastic bone resorption. In addition, complications with bisphosphonate therapy will be reviewed, in particular, osteonecrosis of the jaw associated with bisphosphonate therapy. As survival of myeloma patients increases, therapies to prevent the complications of aggressive myeloma bone disease become more important.

Gene Expression Profiling of Isolated Mesenchymal and Osteoblastic Cells Exhibits a Different Pattern of Expression in Multiple Myeloma Patients as Compared to Healthy Subjects: Potential Relationship with the Presence of Bone Lesions.

Gene expression alterations occurring in the bone microenvironment cells and their potential relationships with the occurrence of bone lesions in multiple myeloma (MM) patients have never been investigated. In this study, we have isolated both mesenchymal (MSC) and osteoblastic (OB) cells, without in vitro differentiation, from bone biopsies obtained by iliac crest of 24 MM patients, 7 MGUS subjects and 8 healthy donors (N) who underwent orthopedics surgery. Bone status was evaluated in all MM patients by total X rays scan and MRI for the spine. Firstly, we evaluated cell proliferation in relationship with growth substrate (bone and glass) and cell phenotype by flow cytometry and immunohistochemistry. We found that both MSC and OB cells have higher cell doubling rate in MM patients as compared to N. Higher expression of alkaline phosphatase and Runx2 was observed in OB as compared to MSC cells in both N and MM patients without osteolytic lesions, but not in osteolytic ones. We performed a gene expression profiling analysis of isolated MSC and OB cells using GeneChip® Affymetrix HG-U133A oligonucleotide arrays. An unsupervised analysis of the most variable genes across the dataset generated a hierarchical clustering with the two major branches containing respectively MSC and OB samples. A multiclass analysis of N, MGUS and MM patients identified 33 differentially expressed probe-set (specific for 27 genes) in MSC cells, and 19 differentially expressed probe-set (13 genes) in OB, and the identified transcripts mainly characterized N versus MM and MGUS samples. A supervised analysis between N and MM samples identified 65 probes (56 genes: 17 up-regulated and 39 down-regulated) differentially expressed in MSC and 35 probes (29 genes, 12 up-regulated and 17 down-regulated) in OB. Notably, genes encoding the Homeobox class proteins, such as HOXB2-6-7, were up-regulated in both MSC and OB of MM patients as compared to N. As regards the bone status, a total of 60 probe-sets (3 up-regulated and 57 down-regulated genes) were found differentially expressed in MSC from osteolytic vs. non-osteolytic MM patients, whereas MGUS-MSC exhibited an intermediate transcriptional profile between osteolytic and non-osteolytic MM patients. A distinct pattern of gene expression profiling was also observed in MSC versus OB when osteolytic and non-osteolytic MM patients were compared (26 vs. 94 differentially expressed probe-sets, respectively), including transcription factors related to MSC osteogenic differentiation belonging to Runx2 pathway (HEY1) or Wnt and BMP signaling On the other hand, few genes were found differentially expressed in OB cells in relationship with the presence of bone lesions. In conclusion, we identified a distinctive transcriptional fingerprint in isolated MSC and OB cells of MM patients as compared to N subjects, which mainly correlated with cell proliferation. Moreover, a different gene expression profile was observed in MSC cells of MM patients according to the presence/absence of bone lesions, highlighting the critical role of the block of the osteogenic differentiation.

11C-choline vs. 18F-FDG PET/CT in assessing bone involvement in patients with multiple myeloma

BackgroundMultiple Myeloma (MM) is a B cell neoplasm causing lytic or osteopenic bone abnormalities. Whole body skeletal survey (WBSS), Magnetic resonance (MR) and 18F-FDG PET/CT are imaging techniques routinely used for the evaluation of bone involvement in MM patients.AimAs MM bone lesions may present low 18F-FDG uptake; the aim of this study was to assess the possible added value and limitations of 11C-Choline to that of 18F-FDG PET/CT in patients affected with MM.MethodsTen patients affected with MM underwent a standard 11C-Choline PET/CT and an 18F-FDG PET/CT within one week. The results of the two scans were compared in terms of number, sites and SUVmax of lesions.ResultsFour patients (40%) had a negative concordant 11C-Choline and 18F-FDG PET/CT scans. Two patients (20%) had a positive 11C-Choline and 18F-FDG PET/CT scans that identified the same number and sites of bone lesions. The remaining four patients (40%) had a positive 11C-Choline and 18F-FDG PET/CT scan, but the two exams identified different number of lesions. Choline showed a mean SUVmax of 5 while FDG showed a mean SUVmax of 3.8 (P = 0.042). Overall, 11C-Choline PET/CT scans detected 37 bone lesions and 18F-FDG PET/CT scans detected 22 bone lesions but the difference was not significant (P = 0.8).ConclusionAccording to these preliminary data, 11C-Choline PET/CT appears to be more sensitive than 18F-FDG PET/CT for the detection of bony myelomatous lesions. If these data are confirmed in larger series of patients, 11C-Choline may be considered a more appropriate functional imaging in association with MRI for MM bone staging.

A prospective comparison of 18F-fluorodeoxyglucose positron emission tomography-computed tomography, magnetic resonance imaging and whole-body planar radiographs in the assessment of bone disease in newly diagnosed multiple myeloma

Bone lesions in multiple myeloma (MM) have been traditionally detected by whole body X-ray (WBXR) survey although magnetic resonance imaging (MRI) has become the gold standard for detecting MM involvement of the spine and pelvis. The aim of this study was to compare a new technique, positron emission tomography (PET) with 18F fluorodeoxyglucose (FDG) integrated with computed tomography (18F-FDG PET-CT), with MRI and WBXR for baseline assessment of bone disease in MM. We prospectively compared 18F-FDG PET-CT, MRI of the spine-pelvis and WBXR in a series of 46 patients with newly diagnosed MM. In 23 patients who received up front autologous transplantation, we also compared post-treatment PET-CT scans with MR images of the spine and pelvis. Overall, PET-CT was superior to planar radiographs in 46% of patients, including 19% with negative WBXR. In 30% of patients, PET-CT scans of the spine and pelvis failed to show abnormal findings in areas in which MRI revealed an abnormal pattern of bone marrow involvement, more frequently of diffuse type. In contrast, in 35% of patients PET-CT enabled the detection of myelomatous lesions in areas which were out of the field of view of MRI. By combining MRI of the spine- pelvis and 18F-FDG PET-CT, the ability to detect sites of active MM, both medullary and extramedullary, was as high as 92%. Following transplantation, 15 patients had negative PET-CT scans (including 13 with a very good partial response or at least a near complete response), but only 8 had normal MRI. MRI of the spine and pelvis still remains the gold standard imaging technique for the detection of bone marrow involvement in MM. 18F-FDG PET-CT provides additional and valuable information for the assessment of myeloma bone disease in areas not covered by MRI.

Significance of macrophage inflammatory protein-1 alpha (MIP-1α) in multiple myeloma

Macrophage inflammatory protein-1 alpha (MIP-1α) is a member of the CC chemokine family and is primarily associated with cell adhesion and migration. It is produced by myeloma (MM) cells and directly stimulates osteoclast formation and differentiation in a dose dependent way. MIP-1α protein levels were elevated in the bone marrow plasma of MM patients and correlated with disease stage and activity. MIP-1α was also elevated in the serum of myeloma patients with severe bone disease and correlated positively with bone resorption markers providing evidence for a causal role of MIP-1α in the development of lytic bone lesions in MM. MIP-1α has also been found to stimulate proliferation, migration and survival of plasma cells. Mice, which were inoculated with myeloma cells and treated with a monoclonal rat anti-mouse MIP-1α antibody, showed a reduction of both paraprotein and lytic lesions. In addition, MIP-1α enhanced adhesive interactions between myeloma and marrow stromal cells, increasing the expression of RANKL and IL-6, which further increased bone destruction and tumor burden. Myeloma patients with high MIP-1α serum levels have poor prognosis. The positive correlation between MIP-1α and β2-microglobulin that has been observed in MM patients at diagnosis further supports the notion that MIP-1α is not only a chemokine with osteoclast activity function but is also implicated in myeloma growth and survival. Therefore, MIP-1α pathway may serve as a target for the development of novel anti-myeloma therapies.

The significance of carboxy-terminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) in assessment of bone disease in patients with multiple myeloma

Bone disease occurring in multiple myeloma is usually evaluated using radiological methods. These methods, however, provide not much information about the dynamic process of bone resorption and formation. This study analysed levels of serum markers of bone turnover (ICTP and OC), reflecting function of osteoclasts and osteoblasts. It demonstrates increased level of ICTP in 75 patients with MM compared to control group (8 persons) and patients with Waldenström's macroglobulinemia (10 persons). The level of ICTP was also higher in patients with more advanced bone disease and probably in higher stage of disease according to Salmon and Durie classification. This tendency was not observed in relation to OC. Result of the research confirms that ICTP may incur sensitive and specific markers of bone lesions in multiple myeloma.

Myeloma Cells Cultivate Megakaryocytes To Promote Lytic Bone Disease.

The development of lytic bone lesions in myeloma (MM) is caused by the activation of osteoclasts through direct and indirect interactions with MM cells and by inactivation of osteoblasts by MM cells. Megakaryocytes have been shown to promote osteoblast differentiation and produce critical factors associated with osteoclast activity. Cells of megakaryocytic linage are often expanded in myelomatous bone. The aim of this study was to investigate the impact of myeloma cells on regulation of bone remodeling by megakaryocytes. Culture of megakaryocytes was prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (>90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. Mesenchymal stem cells (MSCs), osteoclast precursors and CD138-selected MM cells were isolated as previously described (Pittenger et al., Science, 1999; Yaccoby et al., Cancer Res., 2004). Our study revealed that MSCs expressed TPO and that expression of TPO was upregulated 5 fold in MSCs after co-culture with MM cells, as determined by quantitive real time PCR (qRT-PCR). To study the effect on osteoblastogenesis, MSCs were co-cultured with megakaryocytes in the presence and absence of primary MM cells (n=5), in media lacking growth factors. Megakaryocytes promoted osteoblast differentiation as determined by elevated expression of alkaline phosphatase, an effect that was completely abrogated by adding MM cells to the cultures. To study the involvement of megakaryocytes on osteoclastogenesis, we initially examined expression of RANKL and OPG by these cells cultured with and without MM cells, using qRT-PCR. OPG and RANKL were expressed by megakaryocytes and gene expression of both factors was upregulated in co-cultured megakaryocytes. However, whereas OPG was insignificantly upregulated by 1.6±0.3 fold, RANKL expression was increased by 5.1±0.4 folds after co-culture with MM cells (p<0.01). RANKL:OPG expression ratio in cultures of megakaryocytes with and without MM cells were 10.2±2.8 and 2.9±1.6, respectively (p<0.03). This suggests that in the absence of osteoblasts, megakaryocytes are a substantial source of RANKL in myelomatous bone, whereas OPG may serve as a MM cell survival factor by neutralizing TRAIL (Shipman & Croucher, Cancer Res., 2003). The numbers of multinucleated osteoclasts counted after culturing of osteoclast precursors with MM+megakaryocytes, MM cells and megakaryocytes were 453±159, 273±95 and 291±104, respectively (n=3, p<0.04, co-culture vs. MM cells). We conclude that MM cells prevent osteoblast differentiation promoted by megakaryocytes and induce megakaryocytes to increase RANKL:OPG ratio. These effects contribute to the uncoupling processes of osteoclastic resorption and osteoblastic bone formation, induction of lytic bone disease and myeloma progression.

The Role of Whole-Body Magnetic Resonance Imaging in the Staging and Follow-Up of Bone Disease in Multiple Myeloma.

Radiographic skeletal survey (RSS) is the standard diagnostic tool for the screening of bone lesions in multiple myeloma (MM) at diagnosis and during the course of disease. Its major limitation is the low sensitivity in detecting minimal bone lesions and in differentiating active from inactive osteolyses. Several studies demonstrated the superiority of magnetic resonance imaging (MRI) over RSS for the detection of spinal and pelvis bone lesions. However, a significant proportion of patients develops bone lesions elsewhere, therefore MRI of spine and pelvis is inadequate for the staging and follow-up of patients. Recently, whole-body MRI (WB-MRI) has been used with promising results for the detection of secondary bone lesions in patients with non-haematological malignancies. The aims of this study were to evaluate the potential role of WB-MRI in the staging and follow-up of MM patients, using RSS as a standard of reference, and to study the correlation of MRI findings with biochemical markers of bone turnover. Characteristics of the 9 patients included in the study were the following: median age 57 years (46–67), 6 males and 3 females; 4 patients were untreated and asymptomatic, and 5 previously treated with chemotherapy. On the same day, all patients underwent RSS and WB-MRI, and blood sampling for serum osteocalcin (OC) as a marker of bone formation, and for carboxyterminal telopeptide of type I collagen (ICTP) as a marker of bone resorption. RSS and WB-MRI were read by two independent radiologists. WB-MRI was performed with a 1.5-T scanner (Magnetom Symphony Maestro Class). The skull, thorax, pelvis, femoral and lower leg bones were imaged in coronal planes, while sagittal images of the spine were acquired. T1-weighted spinecho (SE) and short-tau inversion time inversion recovery (STIR) sequences (TR 2670, TE 101, TI 150) with a maximum field of view of 450 mm and slice thickness of 5 mm were obtained. As reference standard contrast-enhanced MRI was performed in patients with discordant data at RSS and WB-MRI.RSS was negative in 5 patients, whereas in 4 revealed lytic lesions stable with respect to prior controls. In the group of RSS-negative patients, WB-MRI was positive in 3/5 cases, 2 of whom had marrow and serum progression at the time of evaluation. All 4 RSS-positive patients showed lytic lesions also at WB-MRI with an overlapping pattern of distribution. Only the lesions at humeral bones were not detected by WB-MRI, because humeri are outside the field of view. WB-MRI, however, was superior to RSS in identifying lytic lesions in the spine and pelvis. Biochemical markers of bone metabolism were evaluable in 8/9 cases. ICTP levels were high only in one patient without evidence of bone lesions both at RSS and WB-MRI. OC levels were low in 6 of 8 evaluable patients, and 5 of them had a positive WB-MRI.In conclusion, WB-MRI seems more sensitive than RSS for the detection of bone lesions in MM patients. In particular, it is more suitable for the initial staging of the disease in asymptomatic stage I MM and for the follow-up of patients with a stable picture of lytic lesions at RSS. There is no correlation between ICTP levels and the radiological findings, whereas OC levels are decreased in patients with extensive bone involvement.

Dkk1 Transgenic Mice for the Study of Bone Lesions in Human Multiple Myeloma.

Purpose: Extracellular antagonists of the cellular WNT signalling pathway can be divided into two classes depending on the mechanism by which ligand-receptor interactions are inhibited. Members of the first class including SFRP (secreted frizzled-related proteins), WIF1 (WNT inhibitory factor 1) and CER1 (cerberus homolog 1) bind to WNT proteins in the extracellular milieu. Members of the second class including Dickkopf (DKK) proteins bind to the WNT receptor subunits LRP5/6. Reduced WNT signalling may play a role in the natural history of the bone disease that is a hallmark of multiple myeloma (MM). Gene expression profiling of MM plasma cells has shown that over-expression of the Dickkopf family member DKK1 is linked with abundance and size of osteolytic lesions (N Engl J Med 2003, 349:2483–94). The biological mechanism of DKK1-mediated bone destruction is not known.Experimental procedure: To elucidate the mechanism by which DKK1 facilitates osteolysis, we generated transgenic mice in which mouse Dkk1 is expressed under control of the mouse immunoglobulin Eα enhancer. This enhancer is located 3′ of the immunoglobulin heavy-chain locus and comprised of four individual elements that span ~30 kb of genomic DNA. These elements were subcloned into a contiguous stretch of DNA, which we refer to as “reconstituted” Eα enhancer or HS1234 (Int Immunol 2001, 13:1003–12). Here we appended HS1234 to a Dkk1 cDNA whose expression is controlled by a mouse immunoglobulin VH promoter inserted 5′ of the transcriptional start site of Dkk1. Because of an internal ribosome entry site (IRES) 3′ of Dkk1, the gene encoding enhanced green fluorescence protein (EGFP) is co-expressed with Dkk1; i.e., it is a reporter of Dkk1 expression. A scheme of the structure of the Eα-Dkk1 transgene is depicted in Figure 1. [Display omitted] Results: We generated 14 founder lines of Eα-Dkk1 transgenic mice that expressed Dkk1 and the EGFP-encoding gene by RT-PCR (Figure 2). [Display omitted] The mice contained a variable number of Eα-Dkk1 copies, ranging from 2–3 to more than 100. There was but a loose association of transgene expression (qPCR of Dkk1 mRNA levels) and copy number (qPCR of genomic Ea-Dkk1 fragments); i.e., the high-copy number mice exhibited higher average mRNA levels than the low-copy number mice, although there were outliers in each group. Consistent with Eα-dependent restriction of Dkk1 expression to B/plasma cells, Dkk1 mRNA was readily detectable in lymphoid tissues, such as spleen, mesenteric lymph node and bone marrow, but not in liver.Conclusion: The newly developed Eα-Dkk1 transgenics may provide a valuable model system to elucidate DKK1-dependent bone disease in human MM.

Myeloma cells block RUNX2/CBFA1 activity in human bone marrow osteoblast progenitors and inhibit osteoblast formation and differentiation

AbstractDecreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. In this study, we have investigated the effects of myeloma cells on osteoblast formation and differentiation and the potential role of the critical osteoblast transcription factor RUNX2/CBFA1 (Runt-related transcription factor 2/core-binding factor Runt domain α subunit 1) in the inhibition of osteoblastogenesis in MM. We found that human myeloma cells suppress the formation of human osteoblast progenitors in bone marrow (BM) cultures. Moreover, an inhibitory effect on osteocalcin, alkaline phosphatase, collagen I mRNA, protein expression, and RUNX2/CBFA1 activity by human preosteoblastic cells was observed in cocultures with myeloma cells. The inhibitory effect was more pronounced in the cell-to-cell contact conditions compared with those without the contact and involved the very late antigen 4 (VLA-4) integrin system. Among the soluble osteoblast inhibitors screened, we show the potential contribution of interleukin-7 (IL-7) in the inhibitory effect on osteoblast formation and RUNX2/CBFA1 activity by human myeloma cells in coculture. Finally, our in vitro results were supported in vivo by the finding of a significant reduction in the number of Runx2/Cbfa1-positive cells in the BM biopsies of patients with MM who had osteolytic lesions compared with those who did not have bone lesions, suggesting the critical involvement of RUNX2/CBFA1 in the decreased bone formation in MM. (Blood. 2005;106:2472-2483)

Recommendations for Zoledronic Acid Treatment of Patients with Bone Metastases

The introduction of zoledronic acid, a new-generation bisphosphonate, has greatly extended the use of bisphosphonates in the treatment of patients with bone metastases. On the basis of results from three large, randomized, phase III clinical trials enrolling more than 3,000 patients, zoledronic acid (4 mg via 15-minute infusion) was approved in the United States for the treatment of patients with documented bone metastases from solid tumors in conjunction with standard antineoplastic therapy and patients with multiple myeloma. Zoledronic acid is also approved in Europe for the prevention of skeletal-related events in patients with advanced malignancies involving bone. Current treatment guidelines published by the American Society of Clinical Oncology recommend the use of intravenous bisphosphonates at first radiographic evidence of osteopenia in patients with multiple myeloma or osteolytic bone lesions in patients with breast cancer to significantly reduce the occurrence and delay the onset of skeletal complications. Zoledronic acid has also demonstrated efficacy in the treatment of bone metastases in patients with prostate cancer, lung cancer, and other solid tumors. Bisphosphonate therapy is generally well tolerated but can be associated with increases in serum creatinine. Therefore, monitoring renal function is required for all patients receiving bisphosphonate therapy. Serum creatinine should be monitored before each dose and treatment withheld until any serum creatinine elevations have resolved to baseline levels. Caution should be exercised when treating patients who are receiving other potentially nephrotoxic therapies. With these simple precautions, intravenous bisphosphonate therapy is safe for long-term use and provides durable treatment benefits.

Myeloma Cells Block Runx2/Cbfa1 Activity in Human Bone Marrow Osteoblast Progenitors and Inhibit Osteoblast Formation and Differentiation.

The decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. Runx2/Cbfa1 is a transcription factor highly restricted to the osteoblastic lineage that has a critical role in the osteoblast formation and differentiation. In this study we investigated the potential effect of myeloma cells on osteoblastogenesis and the role of Runx2/Cbfa1 in the decreased bone formation in MM.In a co-culture system performed in presence or absence of a transwell insert with human myeloma cell lines (HMCLs) (RPMI-8226, U266, XG-1, XG-6, OPM-2) and human bone marrow (BM) pre-osteoblastic cells, obtained from BM stromal cells (BMSC), we observed an inhibition of osteocalcin, alkaline phosphatase and collagen I expression at both mRNA and protein level. Consistently we found that the formation of both Colony Forming Unit Fibroblasts (CFU-F) and Colony Forming Unit Osteoblasts (CFU-OB) were suppressed in long term BM culture by several HMCLs. Moreover, by a gel mobility shift assay (EMSA) we found that Runx-2/Cbfa1 activity was significantly inhibited in BMSC/pre-osteoblastic cells after 48 hours of co-cultures with OPM-2 whereas the level of Runx2/Cbfa1 protein was not affected. The inhibition of osteoblast formation, differentiation and the block of the Runx2/Cbfa1 activity in BMSC/pre-osteoblastic was more pronounced in the cell-to-cell contact conditions as compared to those without the cellular contact.Further we evaluated Runx2/Cbfa1 expression by immunohistochemistry in BM biopsies of 16 MM patients finding a significant reduction of the number of Runx2/Cbfa1 positive cells in osteolytic patients as compared to those without bone lesions (median %: 19.93% vs. 38%; p=0.001) that supports the in vitro results.To identify molecules that could contribute to the inhibitory effect of myeloma cells on osteoblast formation, differentiation and Runx2/Cbfa1 activity, we screened HMCLs and fresh purified CD138+ MM cells for the expression of DKK1, IL-7, noggin, gremlin and secreted frizzled-related protein (sFRP)-2, -3, -4 also evaluating the potential involvement of these osteoblast inhibitors. DKK1 mRNA was expressed by 2 out of 5 HMCLs tested and by 8 out of 12 MM patients tested as well as by BMSC/pre-osteoblastic cells and hOB, however soluble DKK-1 at a widely range of concentration had not effect on the number of CFU-F and CFU-OB in human BM cultures and it did not inhibited Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells. Moreover, blocking anti-DKK1 Ab did not blunted the effect of HMCLs on osteoblast formation in human BM cultures. All the HMCLs and fresh CD138+ MM cells tested expressed IL-7 mRNA, as we have previously reported. IL-7 reduced Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells showing an inhibitory effect on CFU-F and CFU-OB formation in BM culture, moreover blocking anti-IL-7 Ab partially reduced the effect of HMCLs in co-cultures. Finally, we found that HMCLs and fresh CD138+ MM cells did not expressed noggin, gremlin, sFRP-2 and rarely produced sFRP-3 and sFRP-4 whereas BMSC/pre-osteoblastic cells and hOB were positive for these factors even if HMCLs had not effect on their expression in co-cultures.In conclusion our data indicate that human myeloma cells inhibit osteoblast formation and differentiation in human BM cultures blocking Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells through the cell-to-cell contact. Soluble factors produced by myeloma cells as IL-7 could contribute to the inhibitory effect on osteoblastogenesis.

Pharmacokinetics (PK) and pharmacodynamics (PD) of AMG 162, a fully human monoclonal antibody to Receptor Activator of NF kappa B Ligand (RANKL), following a single subcutaneous dose to patients with cancer-related bone lesions

8106 Background: RANKL is an essential osteoclastic differentiation/activation factor. AMG 162 PK and PD were evaluated in a phase 1, single dose, randomized, double-blind, double-dummy, active control (pamidronate), dose escalation study, in patients with breast cancer (BC) or multiple myeloma (MM), with lytic or mixed lytic-blastic bone lesions. Methods: Patients (n=54; 3–9/cohort) received a subcutaneous (SC) injection of either AMG 162 (0.1, 0.3, 1.0, or 3.0 mg/kg) or placebo (3:1 ratio) and an intravenous infusion of either 90 mg of pamidronate or saline. Blood and urine samples were collected for determinations of AMG 162 serum concentrations and the bone resorption marker, N-telopeptide levels (uNTx/Cr), respectively. Results: AMG 162 serum levels reached average maximums of 448 ng/mL (0.1 mg/kg) to 19,800 ng/mL (3.0 mg/kg) 14 to 21 days post-dose, and 625 ng/mL (0.1 mg/kg) to 20,100 ng/mL (3.0 mg/kg) 7 to 14 days post-dose in BC and MM patients, respectively. Serum levels were maintained above 100 ng/mL at 0.3 mg/kg and above 3000 ng/mL for 84 days following 3.0 mg/kg in both BC and MM patients. Rapid decreases in uNTx/Cr (within 24 hours) followed AMG 162 administration in BC and MM patients. At 0.3 mg/kg and greater, AMG 162 produced marked suppression in uNTx/Cr with median maximal suppression of greater than 50% in MM and 75% in BC patients. A 1.0 mg/kg dose produced sustained median suppression (>70%) in BC and MM patients for the duration of the study (84 days), while a 3.0 mg/kg dose produced similar suppression in BC yet less suppression in MM patients (∼38%). Conclusions: The favorable PK and PD of AMG 162 potentially allows for infrequent SC dosing in the treatment of bone disorders. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen, Inc. Amgen, Inc. Amgen, Inc.

Expression of receptor activator of NF-kappaB ligand (RANKL) mRNA in human multiple myeloma cells.

Increased bone resorption is a hallmark of multiple myeloma and a result of excessive osteoclast activation. Recently, the receptor activator of NF-kappaB ligand (RANKL) was found to be the critical factor for osteoclastogenesis. Studies showed that myeloma cells induce RANKL expression in bone marrow stromal cells, but it remained a controversy whether myeloma cells directly express RANKL. Therefore, we analyzed the expression of RANKL mRNA in freshly isolated CD138 positive plasma cells from patients with multiple myeloma and osteolytic bone lesions, using three different primer pairs against human RANKL. RANKL mRNA could be detected in bone marrow plasma cells from myeloma patients with osteolytic myeloma bone disease. These findings show that myeloma cells directly express RANKL and indicate that specific blockade of RANKL may be an effective treatment for myeloma bone disease.

Bone lesions in elderly patients with multiple myeloma: a multicenter study by the Society for Geriatric Hematology

Background: Bone lesions in multiple myeloma (MM) have a significant impact on the quality of life of elderly patients, but they have not been extensively investigated in the elderly.Methods: The subjects were 146 elderly MM patients (aged ≥ 65 years, median age 74) admitted to 11 institutions. Bone lesions were compared with those in 65 non‐elderly MM patients.Results: At the time of diagnosis, skeletal symptoms were present in 104 cases and bone pain in 75. Mixed type occurred more often in elderly patients (63.5%) than in controls (28.3%) (P &lt; 0.0001). Lumbar vertebral lesions were more common in elderly than non‐elderly patients (P &lt; 0.0001). Bone lesions restricted physical activity in 71 elderly patients (48.6%). There was a significant difference between elderly patients with and without bone lesions in the rate of detection of plasma cells in bone marrow. Significant differences were detected in serum calcium concentration, the rate of detection of plasma cells in bone marrow, and serum β2‐microglobulin concentration between patients with and without bone pain, and between those with and without fractures. No significant differences were detected in survival time between elderly patients with and without bone lesions, with and without bone pain, or with and without fractures, whereas a significant difference was seen between these subgroups of non‐elderly patients.Conclusion: Although there were no significant differences in the incidence of bone lesions between elderly and non‐elderly patients, the mixed type of bone lesions occurred more often in the elderly than in controls. Lumbar vertebral lesions were more common in elderly than non‐elderly patients. There was no significant difference in prognosis between elderly patients with and those without bone lesions so the treatment strategy for bone lesions in the elderly should be aimed at improving quality of life through direct treatment of the bone lesions, with subsequent improvement of the related symptoms.