Immunohistochemical study of receptor activator of nuclear factor kappa-B ligand (RANK-L) in human osteolytic bone tumors

BACKGROUND AND OBJECTIVES: Osteolytic bone tumors produce intercellular signaling proteins that regulate bone remodeling by altering the rates of osteoclast and osteoblast differentiation and activity. This report examines osteolytic bone tumor expression of receptor activator of nuclear factor B-ligand (RANK-L), a cytokine that is arguably the most critical regulator of osteoclast differentiation and activation. METHODS: This prospective immunohistochemical study examined RANK-L expression in frozen tissues from sixteen surgical specimens of patients who underwent surgery for the treatment of osteolytic bone tumors between 1999 and 2000. RESULTS: RANK-L was positive in 13 of the 16 cases. Primary benign bone tumors, primary malignant bone tumors, and metastasis to bone were positive for RANK-L. CONCLUSIONS: The cells in some, but not all, osteolytic tumors produce the cytokine RANK-L. Further study is necessary to determine in which specific tumors RANK-L is the cytokine responsible for increased osteoclastic activity, and to develop possible therapeutic use of RANK-L antagonists such as osteoprotegerin (OPG).     

Soluble receptor activator of nuclear factor kappaB Fc diminishes prostate cancer progression in bone

Prostate cancer (CaP) develops metastatic bone lesions that consist of a mixture of osteosclerosis and osteolysis. We have previously demonstrated that targeting receptor activator of nuclear factor kappaB ligand (RANKL) with osteoprotegerin (OPG) prevents the osteolytic activity of CaP and its ability to establish tumor in bone. However, OPG can block tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, suggesting that the clinical use of OPG may prevent apoptosis of tumors mediated by TRAIL. Thus, methods to block RANKL activity, other than OPG, may be important. Accordingly, we evaluated the ability of soluble murine RANK-Fc (sRANK-Fc) to prevent progression of established CaP in a severe combined immunodeficient mouse implanted with fetal human bone. We first confirmed that sRANK did not block TRAIL-mediated apoptosis of LuCaP cells in vitro and that it did block LuCaP-conditioned media-induced osteoclastogenesis in vitro. Then, LuCaP 35 CaP cells were injected into the marrow space of the bone implanted in the severe combined immunodeficient mice implanted with fetal human bone and allowed to develop into tumors for 6 weeks. Either vehicle or sRANK-Fc was then administered for 6 weeks. sRANK-Fc diminished tumor-induced osteoblastic lesions as demonstrated by radiograph, bone mineral density measurement, and bone histomorphometry. sRANK-Fc also reduced systemic bone remodeling markers, including serum osteocalcin and bone-specific alkaline phosphatase and urine N-telopeptide of collagen. Finally, sRANK-Fc decreased serum prostate-specific antigen levels and tumor volume in the bone, which indicates decreased tumor burden. In contrast, sRANK-Fc had no effect on s.c. implanted LuCaP cells. We conclude that sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling.     

Survivin在多发性骨髓瘤患者骨髓细胞中的表达及其临床意义

背景与目的:Survivin是凋亡抑制蛋白家族成员之一,在多种恶性肿瘤中高表达,但在正常成人分化组织无表达。其表达与多种肿瘤的预后及肿瘤对放、化疗的耐受有关。本研究旨在通过检测Survivin在正常人、初治及复发/难治多发性骨髓瘤(multiplemyeloma,MM)患者骨髓细胞及骨髓瘤细胞系KM3中的表达,以揭示Survivin在骨髓瘤中的表达与耐药及疗效的关系。方法:RT-PCR和Westernblot法检测29例MM患者(其中初治17例,复发/难治12例)、9例正常人骨髓标本及KM3细胞系中SurvivinmRNA和蛋白质水平表达。结果:骨髓瘤细胞系KM3高表达Survivin。SurvivinmRNA在正常人、初治和难治/复发MM患者骨髓细胞中阳性率分别为22.2%、70.5%和83.3%,表达水平(Survivin/β-actin比值)分别为0.06±0.04、0.31±0.14和0.69±0.24,骨髓瘤患者表达率和表达水平显著高于正常人(P<0.01),而难治/复发MM组表达水平高于初治组(P<0.05)。所有的正常人标本中均未检测到Survivin蛋白,在初治和难治/复发MM组Survivin蛋白阳性率分别为41.1%和58.3%(P>0.05),表达水平(Survivin/α-tubulin半定量比值)分别为0.11±0.07和0.21±0.12,难治/复发MM组明显为高(P<0.05)。17例初治患者中,7例Survivin阳性者4例部分缓解(partialresponse,PR),1例微小反应(minorresponse,MR),2例无变化(nochange,NC),有效率[完全缓解(completeresponse,CR) PR MR]71.4%;10例Survivin阴性者中2例CR,4例PR,2例MR,2例NC,有效率80%,略高于阳性组。但统计学分析未发现两组有效率存在显著性差异。结论:Survivin在骨髓瘤患者中的高表达可能是部分患者对化疗不敏感的原因之一,由于Survivin仅特异性表达于骨髓瘤细胞,Survivin可作为逆转骨髓瘤细胞耐药治疗中的潜在靶点。     

组织因子在多发性骨髓瘤中的表达及其与血管新生的关系

 目的　研究多发性骨髓瘤（MM）患者骨髓组织中组织因子（TF）的表达及其与血管内皮生长因子（VEGF）和骨髓血管新生程度的关系。方法　采用免疫组织化学法对试验组32例初治／复发的MM患者、对照组32例缺铁性贫血患者和健康者骨髓组织的TF、VEGF表达水平和微血管密度（MVD）进行测定。结果　试验组与对照组的TF、VEGF阳性率分别为88 %、78 %;60 %、10 %。试验组与对照组TF、VEGF中位表达水平分别为（++） 、（++）;（-）、（-）;试验组与对照组中位MVD分别为4、0。试验组TF表达水平与血管新生程度等级相关性分析结果显示TF表达水平与血管新生程度呈等级正相关（rs＝0.568,P＜0.001）。结论　MM患者骨髓组织中的TF表达水平与VEGF表达水平高于对照组,与血管新生程度呈等级正相关。     

A study of the biological receptor activator of nuclear factor-kappaB ligand inhibitor, denosumab, in patients with multiple myeloma or bone metastases from breast cancer.

PURPOSE: Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the differentiation, function, and survival of osteoclasts, which play a key role in establishment and propagation of skeletal disease in patients with multiple myeloma or bone metastases as well as many other skeletal diseases. Denosumab (AMG 162), a fully human monoclonal antibody to RANKL, was developed to treat patients with skeletal diseases. EXPERIMENTAL DESIGN: This was a randomized, double-blind, double-dummy, active-controlled, multicenter study to determine the safety and efficacy of denosumab in patients with breast cancer (n = 29) or multiple myeloma (n = 25) with radiologically confirmed bone lesions. Patients received a single dose of either denosumab (0.1, 0.3, 1.0, or 3.0 mg/kg s.c.) or pamidronate (90 mg i.v.). Bone antiresorptive effect was assessed by changes in urinary and serum N-telopeptide levels. Pharmacokinetics of denosumab also were assessed. RESULTS: Following a single s.c. dose of denosumab, levels of urinary and serum N-telopeptide decreased within 1 day, and this decrease lasted through 84 days at the higher denosumab doses. Pamidronate also decreased bone turnover, but the effect diminished progressively through follow-up. Denosumab injections were well tolerated. Mean half-lives of denosumab were 33.3 and 46.3 days for the two highest dosages. CONCLUSIONS: A single s.c. dose of denosumab given to patients with multiple myeloma or bone metastases from breast cancer was well tolerated and reduced bone resorption for at least 84 days. The decrease in bone turnover markers was similar in magnitude but more sustained than with i.v. pamidronate.     

Receptor activator of nuclear factor-kappaB ligand expression by human myeloma cells mediates osteoclast formation in vitro and correlates with bone destruction in vivo

Multiple myeloma (MM) is an incurable B-cell malignancy able to mediate massive destruction of the axial skeleton. The aim of this study was to examine the involvement of the tumor necrosis factor-ligand family member, receptor activator of nuclear factor-kappaB ligand (RANKL), and its naturally occurring antagonist, osteoprotegerin (OPG), in MM biology. Using flow cytometry and two independent anti-RANKL antibodies, we demonstrate RANKL expression in CD38(+++)CD45(+) and CD38(+++)CD45(-) myeloma plasma cell (MPC) subpopulations derived from patients with osteolytic MM. In addition, highly purified subpopulations of MPC express mRNA for both transmembrane and soluble RANKL isoforms but lack expression of OPG mRNA and protein. We also show that RANKL expressed by MPC is functional as in vitro coculture of CD38(+++)CD45(+) and CD38(+++)CD45(-) MPC subpopulations with peripheral blood mononuclear cells resulted in the formation of multinucleate, tartrate-resistant acid phosphatase-positive osteoclasts-like cells capable of forming typical resorption pits. Furthermore, high expression of membrane-associated RANKL by CD38(+++) MPC correlated with the presence of multiple radiological bone lesions in individuals with MM. Together, our data strongly suggest that RANKL expression by MPC confers on them the ability to participate directly in the formation of osteoclast in vivo and extends our knowledge of the involvement of RANKL and OPG in the osteolysis characteristic of this disease.     

Elevated level of plasma basic fibroblast growth factor in multiple myeloma correlates with increased disease activity.

Recent reports that bone marrow angiogenesis is increased in multiple myeloma prompted us to examine plasma concentrations of angiogenic growth factors and to elucidate their clinical and biological significance. In 45 cases including 36 cases of multiple myeloma and 9 cases of monoclonal gammopathies of undetermined significance (MGUS), plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were evaluated. FGF-2 was significantly elevated in 25 out of 45 (56%) of the patients with multiple myeloma compared with control subjects (median 9.01 pg ml vs. 1.58 pg/ml, P < 0.0001). The 25 cases were all active multiple myeloma, and none of the non-active myeloma and MGUS patients showed a high FGF-2 level. VEGF level was also elevated in 26 out of 45 patients (58%) compared with control subjects (median 42.0 pg/ml vs. 15.8 pg/ml, P < 0.0001 for VEGF). VEGF concentration was high in 20 active myelomas, but also in one non-active myeloma and five MGUS. Elevation of FGF-2 level was associated with beta2-microglobulin level, anemia and bone marrow plasma cell percentage, which represent disease activity. Interestingly, none of five Bence-Jones type myelomas, including four clinically active cases, revealed a high plasma FGF-2 level, while all of them showed a high VEGF level. In all five responders, the plasma FGF-2 levels were significantly decreased after chemotherapy. FGF-2 was immunohistochemically detected in the bone marrow myeloma cells of the patients with high plasma FGF-2 level. We conclude that plasma concentration of FGF-2 can be a useful indicator of disease activity.     

Receptor activator of nuclear factor kappaB ligand plays a nonredundant role in doxorubicin-induced apoptosis

Doxorubicin induces apoptosis in a variety of cells. We investigated the expression and function of various tumor necrosis factor (TNF)alpha-homologues and their receptors. CEM cells did not differentially express any one of the TNFalpha-homologous receptors investigated nor TNF-related apoptosis-inducing ligand or TNF-related weakly apoptosis-inducing ligand (TWEAK) in the presence of doxorubicin. In addition to CD95 ligand, however, receptor activator of nuclear factor kappaB ligand (RANKL) was strongly up-regulated. Doxorubicin-induced apoptosis was greatly suppressed in the presence of either neutralizing antibody or RANK-Fc fusion protein. Moreover, neutralizing RANKL also prevented cytochrome c release from mitochondria. RANKL alone was unable to induce significant levels of apoptosis in CEM cells. However, doxorubicin-induced apoptosis was increased >2-fold when exogenous RANKL was added. Therefore, RANKL is necessary but not sufficient to account for early doxorubicin-induced apoptosis in CEM cells. This finding suggests improved chemotherapeutic efficiency of the anthracyclin against susceptible malignant cells in the presence with RANKL.     

Myeloma cells and bone marrow osteoblast interactions: role in the development of osteolytic lesions in multiple myeloma

Bone destruction is the hallmark of multiple myeloma (MM) due to the high capacity of malignant plasma cells to induce a severe imbalance of bone remodeling. Growing evidences suggest that MM cell interactions with bone marrow (BM) osteoblast have a critical role in the pathophysiology of osteolytic lesions. Indeed histomorphometric studies have demonstrated that MM patients with osteolytic bone lesions have lower numbers of osteoblasts and decreased bone formation together with osteoclast activation. Recently, the biological mechanisms involved in the osteoblast inhibition induced by MM cells have begun to be elucidated, underlying the main role of the block of osteoblast differentiation in the development of bone lesions. In this article, we summarize the main mechanisms regulating MM cell and osteoblast interactions.     

多发性骨髓瘤骨髓单个核细胞PTEN mRNA的表达

目的观察PTEN mRNA在多发性骨髓瘤单个核细胞中的表达,探讨不同免疫球蛋白分型的多发性骨髓瘤患者PTEN基因mRNA表达的差异,了解PTEN基因在MM患者中的表达和意义.方法 RT-PCR法扩增多发性骨髓瘤患者单个核细胞中和非肿瘤对照者的PTEN mRNA,以β-actin作为内参照,用图像分析软件测定电泳条带的光密度值,并计算PTEN/β-actin比值.结果多发性骨髓瘤患者单个核细胞PTEN mRNA表达与非肿瘤患者之间差异无显著性.不同免疫球蛋白类型的多发性骨髓瘤患者之间差异无显著性.结论多发性骨髓瘤患者单个核细胞PTEN基因mRNA表达未见异常,其在多发性骨髓瘤中的意义待定.     

Thalidomide downregulates angiogenic genes in bone marrow endothelial cells of patients with active multiple myeloma.

PURPOSE: To study the antiangiogenic effect of thalidomide. PATIENTS AND METHODS: The expression of key angiogenic genes was studied in bone marrow endothelial cells (ECs) of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies unattributed/unassociated (MG[u]), diffuse large B-cell non-Hodgkin's lymphoma, in a Kaposi's sarcoma (KS) cell line, and in healthy human umbilical vein ECs (HUVECs) following exposure to therapeutic doses of thalidomide. RESULTS: Thalidomide markedly downregulates the genes in a dose-dependent fashion in active MMECs and KS cell line, but upregulates them or is ineffective in nonactive MMECs, MG(u)ECs, NHL-ECs, and in HUVECs. Secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor also diminishes according to the dose in culture conditioned media (CM) of active MMECs and KS, whereas it does not change in the other CM. CONCLUSION: Inhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.     

Malignant B-lymphoid cells with bone lesions express receptor activator of nuclear factor-kappaB ligand and vascular endothelial growth factor to enhance osteoclastogenesis.

PURPOSE: Receptor activator of nuclear factor-kappaB ligand (RANKL) is a key mediator of osteoclastogenesis. Because certain types of tumor cells aberrantly express RANKL, and because bone destruction also develops in B-cell lymphomas of bone origin, we investigated RANKL expression and the mechanisms of osteoclastogenesis in B-lymphoid neoplasms. EXPERIMENTAL DESIGN AND RESULTS: Immunohistochemistry of bone specimens resected from patients with primary B-cell lymphoma of bone with bone destruction revealed that lymphoma cells express RANKL as well as vascular endothelial cell growth factor (VEGF). The tumor cells isolated from the bone specimens enhanced osteoclastogenesis in vitro. In contrast, B-cell lymphoma infiltrating to the bone marrow without bone destruction did not express RANKL. Both RANKL and VEGF were expressed by a portion of B-lymphoid cell lines, including Daudi and IM-9. These RANKL-expressing tumor cells enhanced osteoclastogenesis from RAW264.7 cells and human monocyte-derived preosteoclasts in the absence of stromal cells/osteoblasts in a RANKL-dependent manner. Furthermore, conditioned media from Daudi cells enhanced transmigration of preosteoclasts that was inhibited by anti-VEGF antibody, suggesting that tumor cell-derived VEGF mediates recruitment of osteoclast precursors. Moreover, cocultures of B-lymphoid cell lines with osteoclasts enhanced the growth of B-lymphoid cells. CONCLUSIONS: Some malignant B cells aberrantly express functional RANKL as well as VEGF to enhance osteoclastogenesis. The coexpression of RANKL and VEGF may also contribute to the close cellular interactions with osteoclastic cells, thereby forming a vicious cycle between osteoclastic bone destruction and tumor expansion in bone.     

2-甲氧基雌二醇对多发性骨髓瘤患者骨髓细胞的影响

目的:探讨2-甲氧基雌二醇(2ME2)对多发性骨髓瘤患者骨髓瘤细胞及造血前体细胞凋亡的影响。方法:采用DNA片断原位末端标记(TUNEL法)检测多发性骨髓瘤患者骨髓瘤细胞及造血前体细胞凋亡。结果:14例多发性骨髓瘤患者骨髓瘤细胞经1、4、16μmol/L2-甲氧基雌二醇作用48小时后,凋亡率分别为(10.5±1.2)%、(21.2±2.3)%、(36.8±3.6)%,呈现剂量依赖性。经统计学分析,各组之间及与空白对照组相比,差异显著(P<0.05)。骨髓中粒系、红系、淋巴系细胞出现凋亡较少,经统计学分析,各组之间及与空白对照组相比,无明显差异(P>0.05)。相同的2ME2浓度条件下,骨髓中粒系、红系、淋巴系细胞凋亡率与骨髓瘤细胞组凋亡率相比,差异显著(P<0.05)。结论:2-甲氧基雌二醇可选择性诱导骨髓瘤患者骨髓中骨髓瘤细胞凋亡,对骨髓瘤患者骨髓中粒系、红系、淋巴系细胞影响很小。     

Allogeneic bone marrow transplantation in multiple myeloma.

The use of high-dose chemoradiotherapy with allogeneic hemopoietic stem cell support for the treatment of MM began about a decade ago. Because this procedure has been performed increasingly and because larger numbers of patients are being followed for longer periods of time, the proper role of allogeneic BMT in this setting is becoming clearer. Data available thus far indicate that such an approach results in a complete remission rate of at least 50% to 60%, and even higher if applied as consolidation treatment in the remission phase, a transplant-related mortality reported as 40% to 50% and a long-term survival plateau at around 40%. The 40% 5-year probability of relapse-free survival is considerably higher than that observed following autologous BMT and may result from an allogeneic graft-versus-tumor effect (graft versus myeloma) similar to the well-recognized graft-versus-leukemia effect. Although follow-up is still too short to clearly identify the likelihood of cure for MM allotransplant recipients, a certain number of them are currently long-term, disease-free survivors and--we hope--cured. These promising results and the incurability of MM with conventional chemotherapy should, therefore, encourage further application of allogeneic BMT to selected patients with unfavorable prognostic features. Continued efforts to reduce the morbidity and mortality related to the procedure, as well as to design effective pretransplant regimens with lower extramedullary toxicity and to identify those patients most likely to benefit from BMT, will improve the value of allogeneic BMT in MM.     

Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with multiple myeloma.

Multiple myeloma (MM) is a B-cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM). The role of the BM microenvironment in the pathogenesis of the disease has been demonstrated, especially for the survival and growth of the myeloma plasma cells. Functional characterization of the major component of the BM microenvironment, namely the recently characterized mesenchymal stem cells (MSCs), was never performed in MM. Based on a series of 61 consecutive patients, we evaluated the ability of MSCs derived from myeloma patients to differentiate into adipocytes and osteocytes, inhibit T-cell functions, and support normal hematopoiesis. MSCs phenotypic characterization and quantification of interleukin-6 (IL-6) secretion were also performed. As compared to normal MSCs, MSCs from MM patients exhibited normal phenotype, differentiation capacity and long-term hematopoietic support, but showed reduced efficiency to inhibit T-cell proliferation and produced abnormally high amounts of IL-6. Importantly, these characteristics were observed in the absence of any detectable tumor plasma cell. Chromosomal analysis revealed that MM patients MSCs were devoid of chromosomal clonal markers identified in plasma cells. MM MSCs present abnormal features that may participate in the pathogenesis of MM.