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 β2-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.     

Bone marrow angiogenesis in patients with active multiple myeloma.

Factor VIII-related antigen (FVIII-RA)-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM, and monoclonal gammopathies of undetermined significance (MGUS). A five-to sixfold larger area was found in patients with active MM compared to the other two groups. Aquaporin 1 (AQP1)-positive microvessel areas, measured with the same techniques on adjacent tissue sections, were also increased in active MM, and tended to be larger than and closely correlated with the FVIII-RA areas. Numerous mast cells were found in the bone marrow of active MM patients, and counts were strictly correlated with the microvessel density. The conditioned medium (CM) of bone marrow plasma cells from active MM patients stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis, and angiogenesis in vivo (assessed by the chick embryo chorioallantoic membrane [CAM] system) more strongly and frequently than the CM of patients with nonactive MM and MGUS. Immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 46% to 68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data indicate that patients with active MM represent the vascular phase of plasma cell tumors that is induced, at least partly, through FGF-2 and MMP-2. Mast cells possibly contribute to the vascular phase via angiogenic factors in their secretory granules. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells in patients with active MM.     

The Value of the Bone Marrow Plasma Cell Cytoplasmic Light Chain Ratio in Differentiating Between Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

We compared the plasma cell light chain ratios in the bone marrows of 13 patients with multiple myeloma (MM), with those of 13 patients with monoclonal gammopathy of undetermined significance (MGUS). The mean light chain ratio in favour of the paraprotein isotype in the myeloma group was 51.83 (95% confidence limits (CL) 29.52-74.14) while in the MGUS group it was 5.30 (CL 2.07-8.52). The difference between the MGUS and MM groups was significant (p = 0.0005). Neither the bone marrow plasma cell count nor the paraprotein level were significantly correlated with the light chain ratio in either of these two groups. We found a cut-off ratio of 8 to be the most useful in differentiating between myeloma and MGUS. Only one patient with myeloma had a ratio below 8, and one MGUS patient had a ratio above this cut-off point. We conclude that determination of the bone marrow plasma cell light chain ratio is a simple and useful test in differentiating between myeloma and MGUS in difficult cases.     

Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma

Immunohistochemical, cytochemical and ultrastructural data showing vivid angiogenesis and numerous mast cells (MCs) in the bone marrow of 24 patients with active multiple myeloma (MM) compared with 34 patients with non-active MM and 22 patients with monoclonal gammopathy of undetermined significance (MGUS) led us to hypothesize that angiogenesis parallels progression of MM, and that MCs participate in its induction via angiogenic factors in their secretory granules.     

Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma

The oncogene c-maf is translocated in approximately 5%-10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin beta7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin beta7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.     

Multiple myeloma. An update on diagnosis and management

Patients with multiple myeloma must be differentiated from those with monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). The plasma cell labeling index is helpful in differentiating MGUS or SMM from multiple myeloma (MM). No difference in survival was noted between patients given a single alkylating agent and those given a combination of alkylating agents. Alternating cycles of interferon alpha 2 and VBMCP (vincristine, BCNU, melphalan, cyclophosphamide, prednisone) produced a complete or near-complete response in 41% of patients. Allogeneic or syngeneic bone marrow transplantation has produced some benefit. Autologous bone marrow transplantation is potentially applicable to treat more patients. Major problems are eradication of myeloma cells from the bone marrow and removal of myeloma cells from autologous bone marrow. Purging of myeloma cells with monoclonal antibodies and chemotherapy may be helpful. Stem cells from autologous peripheral blood are being used for rescue after high-dose chemotherapy and total-body irradiation.     

Smoldering multiple myeloma and monoclonal gammopathy of undetermined significance

Opinion statement Smoldering multiple myeloma (SMM) consists of the presence of a serum M protein of 30 g/L or more and/or 10% or more bone marrow plasma cells (BMPCs), with no clinical manifestations or symptoms of myeloma. It accounts for approximately 10% of all myelomas, and the median time to progression to a symptomatic multiple myeloma ranges from 2 to 3 years. The main factors for progression are the plasma cell mass (M-protein size and percent of BMPCs), the spinal MRI pattern, the plasma cell proliferative index, and the variant of SMM (“evolving≓ vs “nonevolving≓). Although treatment with thalidomide is promising (based on the results of two phase II trials), outside the context of a clinical trial, a watch-and-wait approach with clinical evaluation every 4 months is recommended until evident symptomatic disease progression occurs. Patients with monoclonal gammopathy of undetermined significance (MGUS) have a serum M protein lower than 30 g/L and a proportion of BMPCs of less than 10%, with no clinical findings or symptoms attributable to the monoclonal gammopathy. MGUS has a high prevalence, and its annual rate of malignant transformation is 1%, such that the actuarial probability of progression to a symptomatic monoclonal gammopathy at 25 years of follow-up is as high as 40%. The factors associated with a higher probability of malignant transformation are a relatively high plasma cell mass, immunoglobulin A M-protein type, and the “evolving≓ variant. It is recommended that patients with MGUS are monitored annually. Importantly, patients with asymptomatic monoclonal gammopathies must not be treated before the development of overt multiple myeloma.     

Management of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM)

Monoclonal gammopathy of undetermined significance (MGUS) is defined as a serum M protein level of less than 3 g/dL, less than 10% clonal plasma cells in the bone marrow, and the absence of end-organ damage. The prevalence of MGUS is 3.2% in the white population but is approximately twice that high in the black population. MGUS may progress to multiple myeloma, AL amyloidosis, Waldenstr?m macroglobulinemia, or lymphoma. The risk of progression is approximately 1% per year, but the risk continues even after more than 25 years of observation. Risk factors for progression include the size of the serum M protein, the type of serum M protein, the number of plasma cells in the bone marrow, and the serum free light chain ratio. Smoldering (asymptomatic) multiple myeloma (SMM) is characterized by the presence of an M protein level of 3 g/dL or higher and/or 10% or more monoclonal plasma cells in the bone marrow but no evidence of end-organ damage. The overall risk of progression to a malignant condition is 10% per year for the first 5 years, approximately 3% per year for the next 5 years, and 1% to 2% per year for the following 10 years. Patients with both MGUS and SMM must be followed up for their lifetime.     

Cytokine and chemokine profiles in multiple myeloma; significance of stromal interaction and correlation of IL-8 production with disease progression

Multiple myeloma (MM) is a product of interactions between tumor plasma cells and multiple cell types native to the bone marrow (BM). We have used antibody array technology to examine the proteins produced by BM stromal cells in response to stimulation by BM taken from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. We observed increased production of the chemokine IL-8 by stromal cells co-cultured with supernatants from bone marrow cells of patients with active myeloma. IL-8 production is correlated with active disease and is dependent upon IL-1beta and NF-kappaB signaling. Consistent with the pro-angiogenic activity of IL-8, increased BM microvessel density (MVD) correlated with stimulation of stromal cell IL-8 production. In addition, the majority of MM cell lines and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1beta induced, and correlates with bone marrow angiogenesis.     

The role of interleukin-1 beta in the pathogenesis of multiple myeloma

Interleukin-1 beta has potent OAF activity, can increase the expression of adhesion molecules, and can induce paracrine IL-6 production (see Fig. 1). These biologic effects of IL-1 beta closely parallel several of the clinical features of human myeloma, such as osteolytic bone lesions, homing of myeloma cells to the bone marrow, and IL-6-induced cell growth. The increased production of adhesion molecules could explain why myeloma cells are found predominantly in the bone marrow. These fixed monoclonal plasma cells could subsequently stimulate osteoclasts through the production of IL-1 beta and paracrine generation of IL-6, resulting in osteolytic disease. Also, IL-6 produced by either a paracrine or autocrine mechanism can support the growth of the myeloma cells that may be manifested clinically by an elevated labeling index. In the future, continued follow-up of IL-1 beta-positive and IL-1 beta-negative MGUS patients should determine whether aberrant expression of IL-1 beta by monoclonal plasma cells is a critical genetic event in the progression of MGUS to myeloma. Because MGUS is relatively common in the general population and myeloma is incurable in almost all cases, identification of MGUS patients who are likely to progress to active myeloma will be important in the development of new therapeutic strategies. For example, an effective chemopreventive agent that prevents or delays the transition from MGUS to myeloma could have a major effect on the treatment of patients with monoclonal gammopathies.     

Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma.     

早期乳腺癌血清VEGF水平与骨髓微转移的相关性及临床意义

目的 检测早期乳腺癌患者血清中VEGF及骨髓标本中CK19的表达水平,探讨二者的相互关系及临床意义.方法 应用ELISA法分别检测6例乳腺纤维腺瘤及64例乳腺癌患者血清中VEGF的水平,同时抽取相应的骨髓血标本,应用实时定量逆转录-聚合酶链反应(qRT-PCR)方法检测标本中CK-19的表达水平,并进行相关性分析.结果 VEGF在乳腺癌患者血清中的表达水平明显高于乳腺纤维腺瘤患者,二者差异显著(P=0.012);在HER2阳性患者中,血清VEGF水平明显高于HER2阴性患者(P=0.016).CK19在乳腺纤维腺瘤患者骨髓标本中未见表达,在乳腺癌患者骨髓血中,阳性表达24例(37.5%),而骨髓中CK19阳性的乳腺癌患者血清VEGF水平显著高于CK19阴性的患者(110.46±90.65 vs 70.88±77.13,P=0.038),且二者的表达呈正相关.结论 早期乳腺癌患者血清VEGF水平与骨髓微转移密切相关,提示VEGF可能参与了肿瘤细胞的侵袭和播散,可作为早期乳腺癌微转移的血清学指标.     

Granulocyte colony stimulating factor-producing multiple myeloma associated with neutrophilia

We report a case of IgG-kappa multiple myeloma associated with neutrophilia (WBC 31,300/microl, neutrophil 90.5%). Interestingly, the serum level of granulocyte colony stimulating factor (G-CSF) in this patient was elevated to 1,500 pg/ml (normal range: 5.78-27.5). Plasma cells were 35% in the bone marrow and were strongly stained with anti-G-CSF antibody. To directly study the production of G-CSF from plasma cells in this patient, CD138 positive plasma cells were purified from bone marrow of multiple myeloma patients by magnetic sorting. The expression of G-CSF mRNA was observed in CD138 positive plasma cells from this myeloma patient with neutrophilia by RT-PCR. In contrast, the expression of G-CSF mRNA was not detected in CD138 positive plasma cells from the other multiple myeloma patients without neutrophilia and 4 human myeloma cell lines (HS-Sultan, IM9, RPMI8226, U266) by RT-PCR. After the CD138 positive plasma cells were cultured in vitro for 48 h, the production of G-CSF protein was confirmed (71.8 pg/ml) in the supernatant by ELISA. These results indicated plasma cells of this myeloma patient directly produced G-CSF and that this was the primary cause of neutrophilia.     

血管新生在急慢性白血病中的研究及临床评价

 目的 观察急慢性白血病患者骨髓血管新生情况,分析血管内皮细胞生长因子（VEGF）及其受体（VEGFR）在急慢性白血病患者中的表达及意义。方法 应用vWF标记免疫组化法观察骨髓血管新生情况,ELISA法检测血浆VEGF水平,流式细胞术（FACS）分析新鲜分离的白血病细胞表面VEG-FR表达。结果 32例初诊白血病患者骨髓微血管密度（MVD）较对照组明显增高,治疗后获缓解者骨髓MVD较治疗前明显降低,与对照组相比差异无统计学意义。32例初诊白血病患者血浆VEGF含量明显高于对照组,化疗后获完全缓解（CR）的急性白血病患者血浆VEGF含量降低至正常水平。31例白血病患者骨髓单个核细胞（BMMNC）27例不同程度表达VEGFR,而5例正常对照骨髓均无VEGFR表达。结论 初诊白血病患者存在骨髓血管新生及血浆VEGF水平增高,白血病细胞不同程度表达VEGFR,阻断VEGF-VEGFR信号通路可成为白血病治疗新的靶点。     

Monoclonal Gammopathy of Undetermined Significance and Smoldering Multiple Myeloma

Monoclonal gammopathy of undetermined significance (MGUS) is characterized by the presence of a serum monoclonal (M) protein level less than 3 g/dL, less than 10% clonal plasma cells in the bone marrow, and the absence of hypercalcemia, renal insufficiency, anemia, or bone lesions attributable to a clonal plasma cell disorder. Patients may be tested for a monoclonal gammopathy by serum protein electrophoresis, immunofixation, and the free light chain (FLC) assay. The prevalence of MGUS is 3% for persons more than 50 years of age and 5% in those more than 70 years of age. The risk of progression to multiple myeloma or a related disorder is 1% per year. The size and type of M protein, the number of bone marrow plasma cells, and the results of the FLC ratio are independent risk factors for progression. Smoldering multiple myeloma (SMM) is a more advanced premalignant phase than MGUS and is characterized by more than 3 g/dL of serum M protein, more than 10% clonal plasma cells in the bone marrow, or both, with no evidence of end-organ damage.     

多发性骨髓瘤患者Survivin、VEGF表达及其相关性

目的:研究多发性骨髓瘤（MM）患者血清及骨髓Survivin、VEGF表达及意义。方法:酶联免疫法（ELASA）检测60例初诊多发性骨髓瘤患者及30例对照组患者血清Survivin、VEGF表达情况;免疫细胞化学法（SABC法）检测骨髓单个核细胞中Survivin、VEGF阳性率。酶联免疫法（ELASA）检测VAD联合沙利度胺治疗4周期后患者血清Survivin、VEGF水平,与治疗前比较。结果:初诊多发性骨髓瘤患者血清Survivin、VEGF表达水平较对照组明显升高;初诊骨髓瘤患者骨髓单个核细胞Survivin、VEGF阳性率较对照组明显提高;经统计学分析,P＜0.05,差异有统计学意义。治疗后患者,血清Survivin、VEGF在不同疗效组有差异,疗效良好组与疗效较差组差异比较,P＜0.05。骨髓中Survivin与VEGF表达阳性率呈正相关,与疾病疗效呈负相关。结论:Survivin及VEGF在多发性骨髓瘤患者中表达增加,二者表达水平与疾病疗效密切相关。二者存在协同关系,可作为评估疗效、预后指标。     

早期乳腺癌血清VEGF水平与骨髓微转移的相关性及临床意义

目的：检测早期乳腺癌患者血清中VEGF及骨髓标本中CK19的表达水平,探讨二者的相互关系及临床意义。方法：应用ELISA法分别检测6例乳腺纤维腺瘤及64例乳腺癌患者血清中VEGF的水平,同时抽取相应的骨髓血标本,应用实时定量逆转录-聚合酶链反应（qRT-PCR）方法检测标本中CK-19的表达水平,并进行相关性分析。结果：VEGF在乳腺癌患者血清中的表达水平明显高于乳腺纤维腺瘤患者,二者差异显著（P=0.012）;在HER2阳性患者中,血清VEGF水平明显高于HER2阴性患者（P=0.016）。CK19在乳腺纤维腺瘤患者骨髓标本中未见表达,在乳腺癌患者骨髓血中,阳性表达24例（37.5%）,而骨髓中CK19阳性的乳腺癌患者血清VEGF水平显著高于CK19阴性的患者（110.46±90.65 vs 70.88±77.13,P=0.038）,且二者的表达呈正相关。结论：早期乳腺癌患者血清VEGF水平与骨髓微转移密切相关,提示VEGF可能参与了肿瘤细胞的侵袭和播散,可作为早期乳腺癌微转移的血清学指标。     

Bone marrow angiogenesis in 400 patients with monoclonal gammopathy of undetermined significance, multiple myeloma, and primary amyloidosis.

PURPOSE: To determine whether bone marrow (BM) angiogenesis progressively increases along the spectrum of plasma cell disorders ranging from monoclonal gammopathy of undetermined significance (MGUS) to advanced myeloma. EXPERIMENTAL DESIGN: Four hundred patients with the following disorders were studied: MGUS (76 patients); smoldering (indolent; early-stage) multiple myeloma (SMM; 112 patients); newly diagnosed, active multiple myeloma (MM; 99 patients); relapsed (advanced) multiple myeloma (RMM; 26 patients); and primary amyloidosis (AL; 87 patients). Forty-two normal control BM samples were studied for comparison. BM angiogenesis was studied in a blinded manner by immunohistochemical staining for CD34 to identify microvessels. RESULTS: The median (range) microvessel density (MVD) per x400 high power field was 1.3 (0-11) in the controls, 1.7 (0-10) in AL, 3 (0-23) in MGUS, 4 (1-30) in SMM, 11 (1-48) in newly diagnosed MM, and 20 (6-47) in RMM; P < 0.001. MVD was significantly higher in MGUS, SMM, newly diagnosed MM, and RMM compared with controls and AL; P < 0.001. MVD was not significantly different between controls and AL. By grading, high-grade angiogenesis was present in 0% of controls and AL, 1% of MGUS, 3% of SMM, 29% of newly diagnosed MM, and 42% of RMM; P < 0.001. MVD correlated with the BM plasma cell labeling index (rho = 0.46, P < 0.001) and BM plasma cell percentage (rho 0.5, P < 0.001). Survival was 28 months in SMM and newly diagnosed MM with high-grade angiogenesis, compared with 53 months for those with low- and intermediate-grade angiogenesis; P = 0.02. CONCLUSIONS: BM angiogenesis progressively increases along the spectrum of plasma cell disorders, from the more benign MGUS stage to advanced myeloma, indicating that angiogenesis may be related to disease progression.     

Comparison of interleukin-6 levels in the bone marrow of multiple myeloma patients with disease severity and clonogenicity in vitro

Fifteen of 25 bone marrow aspirates from 23 patients who presented or had been treated for multiple myeloma at the Royal Marsden Hospital produced myeloma colonies (MY-CFUc) in vitro. There was no correlation between disease severity and the level of interleukin-6 (IL-6) in bone marrow plasma nor was there any evidence that the level of IL-6 was higher in bone marrow aspirates from patients whose tumour produced MY-CFUc in vitro compared with those who did not. The mean level of IL-6 in the whole group of patients was 0.41 ng/ml (range 0.1-0.66 ng/ml), a value similar to that found in plasma from normal donor bone marrow, 0.42 ng/ml (range 0.14-0.62 ng/ml). Separation of peripheral blood cells from serum 24 h after collection, compared with 2 h after collection, resulted in a substantial increase of IL-6 in the serum. The results suggest that levels of IL-6 in bone marrow plasma is not a monitor of disease severity in multiple myeloma (MM) and that the collection and separation of blood and/or bone marrow samples into the cellular and aqueous components should be performed using standardized conditions to minimize inter-sample variation resulting from the release of IL-6 from the cellular components.