Bone lesions and macrophage inflammatory protein-1 alpha (MIP-1a) in human multiple myeloma

Bone lesions are a prominent feature accompanying multiple myeloma. Elucidation of the mechanisms regulating osteolysis is crucial in achieving a good quality of life, as such patients suffer from bone pain even after achieving improvement of the disease by high-dose chemotherapy. Recent research has revealed that bone lysis in myeloma patients is the result of both inhibited bone formation and enhanced bone destruction. It has been considered that bone absorption is regulated by activation of osteoclasts mediated by osteoclast activating factor (OAF) produced from myeloma cells. Macrophage inflammatory protein-1 alpha (MIP-1a) is a member of the chemokine family, and was originally determined as a soluble factor secreted from activated macrophages. Many candidates for OAF had been proposed and MIP-1a is now considered a major OAF. In this review, the significance of MIP-1a in myeloma bone disease is summarized.     

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

Macrophage inflammatory protein-1 alpha (MIP-1alpha) 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-1alpha protein levels were elevated in the bone marrow plasma of MM patients and correlated with disease stage and activity. MIP-1alpha 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-1alpha in the development of lytic bone lesions in MM. MIP-1alpha 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-1alpha antibody, showed a reduction of both paraprotein and lytic lesions. In addition, MIP-1alpha 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-1alpha serum levels have poor prognosis. The positive correlation between MIP-1alpha and beta(2)-microglobulin that has been observed in MM patients at diagnosis further supports the notion that MIP-1alpha is not only a chemokine with osteoclast activity function but is also implicated in myeloma growth and survival. Therefore, MIP-1alpha pathway may serve as a target for the development of novel anti-myeloma therapies.     

多发性骨髓瘤骨病的机制研究及治疗进展

多发性骨髓瘤是血液内科较常见的恶性疾病,其以恶性浆细胞在骨髓中过度增殖和积累为特征,产生大量单克隆免疫球蛋白及其片段,导致终末器官的损害.其中约80％的患者合并有多发性骨髓瘤骨病(MBD),从而严重影响了患者的生活质量及疾病预后.研究发现成骨细胞受抑、破骨细胞激活为其主要发病原因,而多种细胞因子及通路影响了这一机制的发生.本文就MBD发生、发展的最新因素及治疗做一综述.     

细胞融合在多发性骨髓瘤骨病发病机制中的作用探讨

目的 探讨人骨髓基质细胞与人骨髓瘤细胞株RPMI 8226的融合细胞在多发性骨髓瘤骨病发病过程中可能的机制.方法 细胞示踪绿色荧光探针(CMFDA)及红色荧光探针(CMTMR)分另别标记的细胞通过化学促融剂聚乙二醇(PEG-1000)诱导融合,建立细胞融合模型.染色体核型分析,确定融合细胞是否发生细胞核的融合;鉴定融合细胞干性基因及促融相关性基因SIRPα、DC-STAMP的表达情况.结果 PEG-1000能够介导骨髓基质细胞与RPMI 8226细胞融合;超过80％的融合细胞染色体数目在80条左右;融合细胞不仅表现出骨髓基质细胞的特征及干性相关基因c-myc、Klf-4、OCT-4表达阳性,而且融合相关性基因SIRP α及DC-STAMP也表达阳性.结论 骨髓基质细胞与RPMI 8226细胞间可形成融合细胞,融合细胞具有进一步成融的潜力,可能是促进多发性骨髓瘤骨破坏的重要原因之一.     

Bone marrow angiogenesis and progression in multiple myeloma: clinical significance and therapeutic approach

It is now well established that solid tumors depend on angiogenesis. Promoters and inhibitors of angiogenesis are in balance and antiangiogenic strategies aim at repressing the angiogenic process, thus retarding solid tumor progression. Recent data suggest the importance of angiogenesis in hematologic malignancies and several studies reveal an increased angiogenesis in active multiple myeloma. Angiogenesis seems to be a prominent feature of MM progression, and seems to be correlated with the prognosis and the resistance of MM to chemotherapy. Numerous cell populations and cytokines are involved in angiogenesis in multiple myeloma and antiangiogenic therapy with thalidomide is effective in patients with refractory or relapsed disease. The combination of thalidomide and of other immunomodulatory agents with other therapeutic regimens could lead to more effective management of patients with multiple myeloma.     

MUC1在骨髓瘤骨病中的表达及临床意义

目的:探讨骨髓瘤骨病患者的骨髓及外周血中粘蛋白1（mucins1,MUC1）的表达水平与骨髓瘤骨病程度的关系及临床意义。方法:分别取50例骨髓瘤患者（0、1、2、3、4级骨病者各10例）及10例健康对照者的骨髓及外周血,采用实时定量PCR及Western blot方法检测骨髓及外周血中MUC1 mRNA及蛋白的表达。结果:与健康对照组相比,0级、1级、2级、3级、4级骨髓瘤骨病患者骨髓及外周血中MUC1 mRNA及蛋白的表达均明显增高,且升高程度与骨损坏程度呈正相关。结论:MUC1可能参与骨髓瘤骨病的发生发展。     

多发性骨髓瘤骨病患者白细胞介素-17的表达及其意义

 【摘要】　目的　研究多发性骨髓瘤（MM）骨病患者骨髓中白细胞介素17（IL-17）的表达水平及其与骨髓单个核细胞核因子κB受体活化因子配体（RANKL）表达的关系,探讨IL-17在MM骨病发病机制中的作用及其临床意义。方法　采用双抗体夹心酶联免疫吸附（ELISA）法检测33例MM骨病患者及20例对照者骨髓上清中IL-17水平,采用荧光定量PCR检测上述两组骨髓单个核细胞RANKL mRNA的表达。 结果　MM骨病组及对照组的骨髓上清均表达IL-17,骨髓单个核细胞均表达RANKL mRNA。MM骨病组骨髓上清中IL-17的含量［（52.69±4.55）pg／ml］高于对照组［（14.35±1.25）pg／ml］,MM骨病组骨髓单个核细胞RANKL mRNA的表达［（0.96±0.12）pg／ml］高于对照组［（0.42±0.03）pg／ml］,差异均有统计学意义（P＜0.05）。活动期MM骨病患者骨髓IL-17［（76.71±7.06）pg／ml］水平显著高于稳定期MM骨病患者［（40.67±3.84）pg／ml］,差异有统计学意义（P＜0.05）,活动期MM骨病患者骨髓单个核细胞RANKL mRNA的表达水平（1.22±0.27）显著高于稳定期MM骨病患者（0.83±0.12）,差异有统计学意义（P＜0.05）。MM骨病组骨髓IL-17与RANKL的表达呈显著正相关（r＝0.690,P＜0.05）。结论　MM骨病患者骨髓IL-17的表达显著增高,骨髓IL-17水平与MM活动期和（或）稳定期相关,骨髓IL-17与RANKL的表达呈正相关,IL-17可能在MM骨病的发病机制中起重要的作用。     

Elevated circulating sclerostin correlates with advanced disease features and abnormal bone remodeling in symptomatic myeloma: reduction post-bortezomib monotherapy

Sclerostin is a Wingless and Int-1 inhibitor, which is produced by osteocytes and inhibits osteoblast-driven bone formation. Sclerostin is implicated in the pathogenesis of bone loss in metabolic bone disorders but there is no information for its effect on multiple myeloma (MM)-related osteolytic disease. We evaluated circulating sclerostin in 157 newly diagnosed patients with symptomatic myeloma, in 25 with relapsed myeloma who received bortezomib monotherapy, in 21 patients with monoclonal gammopathy of undetermined significance (MGUS), and in 21 healthy controls. Patients with active myeloma had elevated circulating sclerostin compared to MGUS patients and controls (p < 0.01). MM patients who presented with fractures at diagnosis (n = 34) had very high levels of circulating sclerostin compared with all others (p < 0.01), whereas sclerostin correlated negatively with bone specific alkaline phosphatase (a bone formation marker; r = -0.541, p < 0.0001) and positively with C-telopeptide of collagen type-1 (a bone resorption marker; r = 0.524, p < 0.0001). Patients with International Staging System (ISS)-3 disease had higher circulating sclerostin compared to ISS-1 and ISS-2 MM (p = 0.001). Furthermore, patients with high sclerostin (upper quartile, n = 40) had a median survival of 27 months versus 98 months of all others (p = 0.031). Relapsed MM patients had higher levels of circulating sclerostin even compared to newly diagnosed patients (p < 0.01). Bortezomib monotherapy resulted in a reduction of sclerostin by almost 50% in both responders and non-responders. These results suggest that patients with active myeloma have elevated circulating sclerostin, which correlated with advanced disease features including severe bone disease. Our study indicates sclerostin as a possible target for the development of novel therapies to enhance osteoblast function in myeloma.     

Myeloma bone disease: pathophysiology and management.

Bone disease is a major feature of multiple myeloma. Myeloma-induced bone destruction is the result of an increased activity of osteoclasts, which is not accompanied by a comparable increase of osteoblast function. Recent studies have revealed that new molecules such as the receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL), osteoprotegerin (OPG), and macrophage inflammatory protein-1alpha are implicated in osteoclast activation and differentiation, while proteins such as dickkopf-1 inhibit osteoblastic bone formation. These new molecules seem to interfere not only with the biology of myeloma bone destruction but also with tumour growth and survival, creating novel targets for the development of new antimyeloma treatment. Currently, bisphosphonates play a major role in the management of myeloma bone disease. Clodronate, pamidronate and zoledronic acid are the most effective bisphosphonates in symptomatic myeloma patients. Biochemical markers of bone remodeling have been used in an attempt to identify patients more likely to benefit from early treatment with bisphosphonates. Furthermore, using microarray techniques, myeloma patients may be subdivided into molecular subgroups with certain clinical characteristics, such as propensity for lytic lesions that may need early prophylactic treatment. Recent phase I studies with recombinant OPG and monoclonal antibodies to RANKL appear promising.     

多发性骨髓瘤患者骨髓中Treg细胞与预后相关性研究

目的：检测多发性骨髓瘤（Multiple myeloma,MM）患者及正常人外周血和骨髓Treg细胞水平,评价MM患者骨髓微环境的免疫状态,以及免疫状态与疾病水平的相关性。方法采用流式细胞术检测45例MM患者外周血以及骨髓中Treg细胞水平,15例正常人外周血及骨髓中Treg细胞水平。以CD4^＋CD25high^＋细胞占CD4＋细胞的百分比代表Treg细胞水平。结果 MM患者外周血Treg细胞高于正常人外周血,MM患者骨髓中Treg细胞低于正常人骨髓,MM患者骨髓微环境中Treg细胞降低水平与浆细胞比例无关,而与疾病的预后相关,与β2-MG水平及ISS相关。结论 MM骨髓微环境中肿瘤诱导的免疫系统的改变是MM发病的原因之一,MM患者骨髓中Treg细胞降低比例与预后相关,而与肿瘤负担无关。     

Quantification of bone marrow plasma cell infiltration in multiple myeloma: usefulness of bone marrow aspirate clot with CD138 immunohistochemistry

Accurate quantification of plasma cells (PCs) in bone marrow (BM) is critical for diagnosis and assessment of treatment response in patients with multiple myeloma (MM). We compared the % of BM PC quantified by 250 cell differential count on May–Giemsa‐stained BM smears, by counting 500 – 2500 cells in 2 – 5 representative microscopy fields in CD138‐immunostained BM clot and biopsy sections, and CD38/CD45/CD138 gated BM PCs on flow cytometry (FCM) in 150 sets of BM samples from 120 patients. Percentages of PC were significantly correlated between BM biopsy and clot, and between smear and FCM (r = 0.96, 0.93, respectively). However, quantification by smear and FCM significantly underestimated the PC compared to biopsy or clot, and the degree of underestimation increased with blood dilution. FCM consistently showed lower % of PC compared to aspirate smears. Fifty‐nine of 103 patients with M‐protein level < 3000 mg/dL in serum or 500 mg/24 h in urine and diagnosed with monoclonal gammopathy of undetermined significance (MGUS) based on smear alone were reclassified as smoldering MM when reassessed using CD138‐stained biopsy/clot sections. Among the 72 patients with sMM diagnosed by BM biopsy and/clot, three patients (4.2%) had extensive BM infiltration of PC (≥ 60%) and required treatment. Our data clearly showed the necessity of CD138 immunostaining of BM biopsy/clot specimens for correct diagnosis of MM and related disorders. Copyright © 2016 John Wiley & Sons, Ltd.     

Correlation of sera TNF-alpha with percentage of bone marrow plasma cells,LDH, beta2-microglobulin,and clinical stage in multiple myeloma

Tumor necrosis factor-α (TNF-α) is important for function, differentiation, and transformation of B-lymphocytes in multiple myeloma (MM) but can also induce apoptosis of myeloma cells. Based on this opposite effect, it is very crucial to analyze the correlation of the serum level of TNF-α with clinical parameters of the patients. In this article, we analyzed 18 MM patients, 48% male and 52% female, with a mean age of 52 yr (range: 35–81 yr), clinical stage I in 21.4%, stage II in 26.4%, and stage III in 52.2% of patients. Patients with advanced clinical stage, presence of osteolysis, and elevated lactate dehydrogenase (LDH) had a significant difference (Mann-Whitney U-test, p<0.05) in the serum level of TNF-α in comparison with those in the early stage, without osteolysis, and normal LDH. The correlation of individual values of TNF-α with the percentage of plasma cells in the bone marrow, LDH, β₂-microglobulin, fibrinogen, and sedimentation rate was significant (p<0.05). However, we have not found a significant correlation between TNF-α and concentration of hemoglobin, the number of white blood cells or platelets (p>0.05). We concluded that our data indicate determination of TNF-α as a good parameter for estimation of tumor mass presence, among individual patients with MM, and may by used for monitoring during application of different therapy protocols.     

Serum concentrations of Dickkopf-1 protein are increased in patients with multiple myeloma and reduced after autologous stem cell transplantation

Dickkopf-1 (DKK-1) protein, a soluble inhibitor of Wnt signalling, has been implicated in the pathogenesis of myeloma bone disease through the suppression of osteoblast differentiation. In this study, serum concentrations of DKK-1 were measured in 50 myeloma patients (32 at diagnosis and 18 before and after autologous stem cell transplantation (ASCT), 18 patients with monoclonal gammopathy of undetermined significance (MGUS), and 22 healthy controls. Serum DKK-1 levels were increased in MM at diagnosis compared with MGUS (mean +/- SD: 67 +/- 54 ng/mL vs. 38 +/- 13 ng/mL; p = 0.006) and controls (31 +/- 11 ng/mL; p = 0.02), while there was no difference between MGUS patients and controls. Although patients with stage 2 and 3 myeloma had higher DKK-1 values than stage 1 patients (79 +/- 63 vs. 40 +/- 13; p = 0.005), no significant correlation between serum DKK-1 and myeloma bone disease was observed. Myeloma patients before ASCT also had increased levels of DKK-1 (63 +/- 77 ng/mL; p = 0.03) compared with controls, supporting the notion that DKK-1 may be responsible for the suppressed osteoblast activity even in patients with low tumor burden. After ASCT, there was a sustained decrease in DKK-1 levels over time, while bone formation markers elevated, suggesting that the reduction of DKK-1 levels after ASCT may correlate with the normalization of osteoblast function. These results could provide the basis for developing agents that block DKK-1, thus restoring osteoblast function and counteracting the increased osteoclastogenesis observed in myeloma.     

Genomic aberrations in anaplastic multiple myeloma: High frequency of 1q21(CKS1B) amplifications

Anaplastic multiple myeloma (AMM) is a rare morphologic variant of MM with adverse prognosis. The underlying molecular cytogenetic abnormalities are poorly understood. We investigated 11 patients with AMM for myeloma associated cytogenetic aberrations and compared with 188 non-anaplastic MM using fluorescent in situ hybridization. Of the 11 AMM patients studied, 10 had CKS1B amplification, 5 hemizygous 17p(p53) deletions, 4 13q14 deletions, 4 t(4:14), and 2 had t(11:14). AMM was associated with significantly higher prevalence of CKS1B amplification (91% vs. 34%, p < 0.001), 17p(p53) deletion (45% vs. 11%, p = 0.006) and t(4,14) (36% vs. 14%, p = 0.015) than non-anaplastic MM, which may have resulted in the genetic instability and more aggressive clinical course.     

Flow cytometric differentiation of abnormal and normal plasma cells in the bone marrow in patients with multiple myeloma and its precursor diseases

Flow cytometric (FC) enumeration of abnormal plasma cells (APCs) for diagnosis and prognostication of plasma cell dyscrasias (PCD) is challenging. We studied antigen expression in normal plasma cells (NPC) (N = 34) and APC in a series of unselected PCD (N = 59). NPC subpopulations often demonstrated CD19(−), CD20(+), CD45(−) or dim and CD56(+), an immunophenotype observed in PCD. However abnormal CD81 was only observed in APCs (APC detection sensitivity 95%; specificity 100%). We evaluated differences in antigen expression patterns among MGUS (N = 14), SMM (N = 35) and MM (N = 10), finding the combination of CD45 and CD56 helpful in differentiating MGUS from SMM and MM (p = 0.0002).     

Estrogen sulfotransferase (SULT1E1) expression in benign and malignant human bone tumors

BACKGROUND AND OBJECTIVES: 17beta-estradiol regulates growth and differentiation in normal and malignant bone. E2 is inactivated to 17beta-estradiol-sulfate through estrogen sulfotransferase (SULT1E1). RESULTS: In an explorative study, SULT1E1 mRNA expression was assessed in a broad range of samples from benign, primary and secondary malignant bone tumors. We detected SULT1E1 mRNA in 31/50 tumor samples (10/19 malignant, 6/13 benign tumors; 15/18 metastases). Significantly more SULT1E1-positive samples were found in metastases than in primary bone tumors (P = 0.019). Yet, there was no difference between malignant and benign primary tumors (P = 0.718). SULT1E1 mRNA levels were not related to patients' age, gender, tumor location, stage, grading, and chemotherapy pretreatment. Relative SULT1E1 mRNA levels did not correlate with that of estrogen-receptor alpha (ERalpha) as assessed by quantitative TaqMan PCR (10 malignant, 8 benign tissue samples). In the latter, ERalpha mRNA, but not SULT1E1 mRNA levels were significantly lower than in the malignant samples (P = 0.006 and P = 0.71, respectively). Also, pronounced expression of SULT1E1 mRNA but not of ERalpha mRNA was observed in osteosarcoma (MG-63, HOS) and Ewing's sarcoma (TC-71) cells, while human osteoblasts and BMSC contained ERalpha but not SULT1E1 mRNA. CONCLUSION: Frequent expression of SULT1E1 mRNA in various human bone tumors suggests that sulfonation might be important to control E2 levels and activity.     

Aggressive angiofollicular lymph node hyperplasia (Castleman's disease) treated with high dose melphalan and autologous bone marrow transplantation

A case of aggressive widespread angiofollicular lymph node hyperplasia in a 42-year-old male, treated with high dose melphalan is presented. The disease had failed to enter a durable remission after chemotherapy. High dose melphalan with autologous bone marrow transplantation achieved a complete remission which has lasted for 15 months to date. This approach can be considered when other measures fail.     

MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features.     

Expression levels of IL-27 and IL-17 in multiple myeloma patients: A higher ratio of IL-27:IL-17 in bone marrow was associated with a superior progression-free survival

The goal of the study was to investigate the levels of interleukin-27 (IL-27) and IL-17 in bone marrow (BM) and peripheral blood (PB) of multiple myeloma (MM). The levels of IL-27 and IL-17 were determined in MM patients and controls using ELISA. The results showed a decreased IL-27 and elevated IL-17 level in MM patients and a negative association of IL-27 with IL-17. The ratio of IL-27:IL-17 in BM of newly diagnosed MM was significantly decreased and correlated with the progression of disease. Multivariate analysis showed that a higher ratio of IL-27:IL-17 in BM was associated with a superior progression-free survival (HR = 0.160; 95% CI: 0.058–0.443; p < 0.001). Our results suggest that there might be a possible competitive role of IL-27 and IL-17 in MM.