In vitro Ig-synthesis and proliferative activity in multiple myeloma are stimulated by different growth factors

Plasma cells isolated from bone marrow (BM) aspirates of 15 patients with active multiple myeloma (MM) were cultured and analysed for in vitro proliferative response and Ig-synthesis upon stimulation with interleukin-3 (IL-3), interleukin-4 (IL-4) and interleukin-6 (IL-6). The proliferative response, determined as Ki-67 positivity in concentrated plasma cells, was increased by IL-6 (Stimulation Index, SI = 1.77 +/- 0.21 (M +/- SEM] but not by IL-3 or IL-4. This proliferation could be blocked by anti-IL-6. In vitro Ig-synthesis was stimulated by IL-4 (SI = 1.62 +/- 0.12 (M +/- SEM) P less than 0.05) but not by IL-6 or IL-3. This effect was not antagonized by anti-IL-6. An inverse correlation was found in this group of patients between the IL-6 induced stimulation of plasma cell proliferative activity and the IL-4 induced increase of Ig-synthesis (P = 0.027). These data indicate in MM that Ig-synthesis and the in vitro proliferative activity may be stimulated by different haematopoietic growth factors, which may reflect the involvement of different responding cells.     

Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P <.001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P =.02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P =.002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.     

Granulocyte-macrophage colony-stimulating factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the growth of multiple myeloma (MM) was investigated in 21 patients with MM. In 17 patients with proliferating myeloma cells in vivo, recombinant GM-CSF significantly increased the endogenous-IL-6-mediated spontaneous myeloma cell proliferation occurring in 5-day cultures of tumor cells in vitro (P less than .01). Furthermore, GM-CSF was detected in 5-day culture supernatants of myeloma bone marrow cells. This endogenous GM-CSF was produced by the myeloma bone marrow microenvironment but not by myeloma cells and contributed to the spontaneous myeloma-cell proliferation observed in 5-day cultures. In fact, this proliferation was partially blocked (67%) by anti-GM-CSF monoclonal antibodies. The stimulatory effect of rGM-CSF was mediated through IL-6 because it was abrogated by anti-IL-6 monoclonal antibodies. rGM-CSF did not reproducibly increase the endogenous IL-6 production in short-term cultures of bone marrow cells of MM patients. Using an IL-6-dependent myeloma cell line (XG-1 cell line), rGM-CSF was shown to act directly on myeloma cells stimulating by twofold their IL-6 responsiveness. rGM-CSF did not induce any IL-6 production in XG-1 cells, nor was it able to sustain their growth alone. Although no detectable GM-CSF levels were found in the peripheral or bone marrow blood of MM patients, it is possible that GM-CSF, produced locally by the tumoral environment, enhances the IL-6 responsiveness of myeloma cells in vivo in a way similar to that reported here in vitro.     

Immunocytochemical evaluation of the percentage of proliferating cells in pathological bone marrow and peripheral blood samples with the Ki-67 and anti-bromo-deoxyuridine monoclonal antibodies

The monoclonal antibody Ki-67, directed against a nuclear antigen expressed by dividing cells in all the phases of cell cycle except G0 and early G1, was used in combination with an anti-BrdU monoclonal antibody, reacting selectively with cells in S-phase, for assessing the percentage of proliferating cells in bone marrow and peripheral blood samples from patients with lymphoma, leukaemia and multiple myeloma. Immunocytochemical labelling of proliferating cells was performed on marrow frozen sections and/or cytospins using an immunoalkaline phosphatase (APAAP) technique that made it possible to obtain proliferative index measurements in a few hours in contrast to the 3-7 d needed with tritiated thymidine. In the 54 marrow lymphoma cases studied a highly significant correlation was observed between the proportion of Ki-67 (+) cells and the separation into low- and high-grade malignant lymphomas according to the Kiel classification. In patients with multiple myeloma at the first diagnosis, the percentage of Ki-67 (+) cells was low (6-10%). In contrast, a high percentage of Ki-67 (+) cells (40-50%) was observed in a young adult with multiple myeloma, in a patient who first presented at the clinical observation with an extradural mass and in three patients who developed extramedullary masses several years after the initial diagnosis of myeloma. In acute lymphoblastic leukaemias of common type the mean value of Ki-67 labelling was 31.3%. Because of their simplicity and rapidity, immunocytochemical techniques may be expected to replace autoradiography and flow cytometry for the detection of proliferating cells in haematological samples.     

Monoclonal antibody Ki-67 as a marker of proliferative activity in monoclonal gammopathies

In 16 patients with monoclonal gammopathies of undetermined significance (MGUS) and in 49 with multiple myeloma (MM, 43 untreated and 6 relapsed) we used immunocytochemistry to determine the percentages of bone marrow plasma cells (BMPC) that incorporate bromodeoxyuridine (BUDR-labeling index, BUDR-LI) in vitro and that label with the monoclonal antibody Ki-67 (which recognizes an antigen thought to identify the growth fraction of the population, Ki-67 GF). Both mean and range values were greater for Ki-67 GF than for BUDR-LI. Most patients with high Ki-67 GF also had high BUDR-LI, although a linear correlation was not found between the two parameters. MGUS has lower values than MM, and the difference was much greater for Ki-67 GF than for BUDR-LI (p less than 0.005 vs. p less than 0.05). Differences in Ki-67 GF but not in BUDR-LI were found between MGUS and stage I MM (p less than 0.0005) and between grouped stage I and II MM and stage III MM (p less than 0.025). Both Ki-67 GF and BUDR-LI were significantly (p less than 0.005) greater in relapsed than in untreated MM. Determining Ki-67 GF as a proliferative parameter could be a better way of studying the kinetics of (BMPC) in MGUS and MM than determining the BUDR-LI, since a wider range of values is obtained and this allows patient groups with different clinical characteristics to be separated more easily.     

多发性骨髓瘤血管内皮生长因子和白细胞介素-6相互作用的研究

目的　研究人类多发性骨髓瘤(MM)细胞系和MM患者骨髓基质细胞(BMSCs)之间相互作用对血管内皮生长因子(VEGF)和IL6分泌的调控作用,分析VEGF和IL6的相互作用在MM发病机制中的意义。方法　建立MMBMSCs和正常人BMSCs(NBMSCs)的培养体系,用IL6、抗IL6抗体、VEGF、抗VEGF抗体作用于BMSCs和(或)MM细胞系U266后,ELISA方法检测其VEGF和IL6的分泌量。结果　U266分泌VEGF,但不分泌IL6,而MMBMSCs和NBMSCs既分泌VEGF又分泌IL6。重组人VEGF刺激BMSCs后,以时间和剂量依赖性的方式诱导IL6的分泌,此效应可被抗VEGF抗体抑制。外源性IL6促进BMSCs分泌VEGF。当U266与BMSCs黏附后,VEGF分泌增加25~50倍,IL6增加55~90倍,两者差异有统计学意义(P<005);分别加入抗VEGF或抗IL6抗体,IL6或VEGF的分泌受抑。重组人IL6作用于U266,可诱导剂量依赖性的VEGF分泌的增加,此反应可被抗IL6抗体抑制。结论　在MM中,MM细胞和BMSCs之间的相互作用调节VEGF和IL6的分泌,促进MM细胞的生长和血管新生,在MM的发病机制中发挥重要作用,为针对骨髓微环境的靶位治疗提供了理论依据。     

Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P <.001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P <.05) and to a variable degree (up to 5.3-fold; P <.005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6. (Blood. 2000;95:2630-2636)     

Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization

Interphase fluorescence in situ hybridization (FISH) studies of chromosomal region 13q14 were performed to investigate the incidence and clinical importance of deletions in multiple myeloma (MM). Monoallelic deletions of the retinoblastoma-1 (rb-1) gene and the D13S319 locus were observed in 48 of 104 patients (46.2%) and in 28 of 72 (38.9%) patients, respectively, with newly diagnosed MM. FISH studies found that 13q14 was deleted in all 17 patients with karyotypic evidence of monosomy 13 or deletion of 13q but also in 9 of 19 patients with apparently normal karyotypes. Patients with a 13q14 deletion were more likely to have stage III disease (P =.022), higher serum levels of beta(2)-microglobulin (P =.059), and a higher percentage of bone marrow plasma cells (P =.085) than patients with a normal 13q14 status on FISH analysis. In patients with a deletion of 13q14, myeloma cell proliferation (Ki-67) was markedly increased (22.0% +/- 6.9% compared with 15.6% +/- 8.2% in patients without the deletion; P =.0008). Evaluation of bromodeoxyuridine incorporation in 5 patients revealed that both rb-1-deleted and rb-1-normal MM subpopulations were proliferative. The presence of a 13q14 deletion on FISH analysis was associated with a significantly lower rate of response to conventional-dose chemotherapy (40.8% compared with 78. 6%; P =.009) and a shorter overall survival (24.2 months compared with > 60 months; P <.005) than in patients without the deletion. Multivariate analysis of prognostic factors confirmed the independent predictive value of 13q14 deletions for shortened survival. In conclusion, deletions of 13q14 are frequently detected by interphase FISH in patients with newly diagnosed MM, correlate with increased proliferative activity, and represent an independent adverse prognostic feature in MM. (Blood. 2000;95:1925-1930)     

MPC-1-CD49e- immature myeloma cells include CD45+ subpopulations that can proliferate in response to IL-6 in human myelomas

Using three-colour phenotypic analysis, we detected five subpopulations of myeloma cells (CD38++) in the bone marrow mononuclear cells of human myeloma patients: MPC-1-CD45-CD49e-, MPC-1-CD45+CD49e-, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- and MPC-1+CD45+CD49e+. Most of the myeloma cells did not express CD45 but a few MPC-1- immature myeloma cells and some MPC-1+ myeloma cells expressed CD45 and CD45RO but not CD45RA, whereas all of normal early plasma cells in the peripheral blood, lymph node plasma cells and bone marrow plasma cells expressed CD45 and CD45RA, CD45RB but not CD45RO. In order to clarify the biological character of these myeloma subpopulations, we examined the expression of Ki-67 antigen. Proliferating myeloma cells (Ki-67+) were found in the MPC-1- fractions and the MPC-1-CD45+ fractions rather than MPC-1-CD45- fractions. Next, in order to further clarify the biological difference of two immature subpopulations (MPC-1-CD45-CD49e- and MPC-1- CD45+CD49e-), determined cell viability and phenotypic change after culturing with interleukin 6 (IL-6) in vitro. In the presence of IL-6, MPC-1-CD45+ cells kept their viability more than MPC-1-CD45- cells and some MPC-1-CD45- cells could be converted to MPC-1-CD45+ cells. In conclusion, these data suggest that human myeloma cells are phenotypically subdivided into five subpopulations, and among these subpopulations MPC-1-CD45+CD49e- but not MPC-1-CD45-CD49e- immature cells contain proliferating cells in response to IL-6, and IL-6 can also induce expression of CD45 on MPC-1-CD45- subpopulation of immature myeloma cells.     

CD40 ligand triggered interleukin-6 secretion in multiple myeloma

Previous studies have suggested that interleukin-6 (IL-6) may mediate growth of multiple myeloma (MM) in either an autocrine or paracrine growth mechanism. However, those molecules which can trigger IL-6 secretion either by tumor cells or non-MM marrow cells are not well characterized. In the present study, we have examined the expression and functional significance of CD40 on MM and plasma cell leukemia (PCL) cells and derived cell lines, as well as long-term bone marrow stromal cells (BMSCs) and derived cell lines. CD40 was expressed on the majority of MM cells (> 90%) and BMSCs (> 70%). Triggering via CD40 using NIH3T3 CD40 ligand transfectant (CD40LT) cells increased (> 30%) cell surface CD80, CD18, CD11a, CD11b, and CD11c expression on MM cell lines. Culture with either fresh or paraformaldehyde fixed NIH3T3 CD40LT cells upregulates IL-6 secretion in MM cells and MM-derived cell lines, as well as normal and MM bone marrow mononuclear cells (BMMCs), BMSCs, and BMSC lines; this effect can be specifically blocked by anti- CD40 monoclonal antibody (MoAb). BMMCs and BMSCs from patients with MM secreted significantly more IL-6 than those from healthy donors (n = 3, P < .001); moreover, after stimulation using CD40L, IL-6 secretion was fourfold greater (n = 3, P < .001) from MM BMMCs and BMSCs than from normal BMMCs and BMSCs. Myeloma (CD38+CD45RA-) cells and non-MM (CD38+CD45RA+, CD38-CD45RA+, and CD38-CD45RA-) BMMCs were separated by dual fluorescence cell sorting. The latter secreted fourfold more IL-6 than the former (n = 2, P < .001). Increased IL-6 secretion (up to 28- fold) and proliferation (Stimulation index 10) by CD38+CD45RA-MM cells was triggered by culture with NIH3T3 CD40LT cells. Finally, anti- CD40MoAb partially (30%) blocked tumor cell to BMSC adhesion-induced IL- 6 secretion. These studies support the view that CD40L may trigger IL-6 secretion by both MM cells and BMSCs and that IL-6-mediated autocrine and paracrine growth mechanisms may be possible in MM.     

Syndecan-1 and angiogenic cytokines in multiple myeloma: correlation with bone marrow angiogenesis and survival

Angiogenesis is a complex process involved in the proliferation and metastasis of malignant tumours, and partly triggered by the secretion of various angiogenic factors by tumour cells or cells in the stromal environment. We investigated the correlation between bone marrow angiogenesis, estimated as microvessel density (MVD), and interleukin-6 (IL-6), basic fibroblastic growth factor (bFGF), hepatocyte growth factor (HGF) and syndecan-1 in 67 patients with newly diagnosed multiple myeloma, and evaluated the prognostic value of these parameters. Circulating levels of IL-6, bFGF, HGF and syndecan-1 were significantly higher in patients than in controls. Moreover, in patients, bone marrow levels of bFGF, HGF and syndecan-1 were higher than peripheral blood levels. Positive correlations were found between MVD and syndecan-1 blood levels (r = 0.33, P = 0.017), syndecan-1 bone marrow levels (r = 0.49, P = 0.046) and HGF blood levels (r = 0.36, P = 0.008) respectively. High MVD and high blood levels of IL-6, HGF and syndecan-1 were predictive of a shorter survival. In a multivariate survival analysis MVD and blood levels of IL-6 retained independent prognostic significance, while in a survival analysis without MVD the peripheral blood levels of HGF and syndecan-1 were strong independent prognostic factors.     

Incidence of apoptosis and cell proliferation in multiple myeloma: correlation with bcl-2 protein expression and serum levels of interleukin-6 (IL-6) and soluble IL-6 receptor

We evaluated the in vivo incidence of apoptosis and cell proliferation in multiple myeloma (MM) and investigated the correlation of both cellular events with histological tumour stage and grade, bcl-2 protein expression, serum IL-6 and sIL-6R. MATERIAL AND METHODS: The TUNEL method was used to assess apoptosis and immunohistochemistry to assess the expression of proliferating cell nuclear antigen (PCNA) and bcl-2 protein in 30 bone marrow biopsy specimens. The apoptotic index (AI) and proliferative index (PI) were defined as the percentage of TUNEL and PCNA positive plasma cells, respectively. RESULTS: The mean AI was 0.162% and the mean PI 27.44%. A positive correlation between AI and PI was found (r = 0.44, P = 0.017). PI was also correlated with tumour grade (P = 0.015). The mean bcl-2 protein expression was 70% and did not correlate with AI or PI, but was higher in specimens taken at first diagnosis than in specimens taken after response to treatment (P = 0.035). The mean serum IL-6 and sIL-6R values were 9.43 pg mL-1 and 47.27 ng mL-1, respectively. These parameters did not correlate with AI or PI. CONCLUSIONS: The results indicate that MM might be among the malignancies with very low incidence of apoptosis. Proliferative activity increased in parallel with tumour histological grade. A positive correlation between apoptosis and proliferation was observed, but the incidence of these two cellular events seems not to be related to the bcl-2 protein expression and the serum levels of IL-6 and sIL-6R.     

Tc-99m methoxyisobutylisonitrile bone marrow imaging for predicting the levels of myeloma cells in bone marrow in multiple myeloma: correlation with CD38/CD138 expressing myeloma cells

The percentage of myeloma cells in bone marrow is subsequently an important index of disease in patients with multiple myeloma (MM). Bone marrow myeloma cells can be detected by strong CD38/CD138 positivity and light scatter characteristics using flow cytometry. The aim of the study was to evaluate the relationship between the degree of Tc-99m methoxyisobutylisonitrile (MIBI) uptake and the percentage of CD38/CD138 expressing myeloma cells in the bone marrow of patients with MM. A total of 15 patients with MM (mean age: 61.7+/-2.4 years; 7 F and 8 M) were included in the study. Tc-99m MIBI imaging was obtained 20 min after injection of 740 MBq Tc-99m MIBI. Planar spot images of the pelvis and thorax were acquired. The uptake of Tc-99m MIBI in the bone marrow was evaluated using a qualitative and also a semiquantitative scoring system for the bone marrow in areas that included the proximal femurs, anterior iliac crest, and sternum. In all patients, flow cytometry was performed for assessing the percentage of CD38/CD138 expressing myeloma cells in the bone marrow samples. There was a statistically significant positive correlation between the percentage of CD38/CD138 expressing plasma cells in bone marrow and both mean qualitative (r=0.689, p=0.005) and semiquantitative (r=0.669, p=0.006) results of Tc-99m MIBI uptake. In conclusion, our results indicate that increased Tc-99m MIBI uptake of bone marrow is related to the percentage of plasma cell infiltration of bone marrow. Tc-99m MIBI bone marrow imaging may be a useful tool for predicting the levels of myeloma cells in bone marrow of patients with MM.     

Plasma cell growth fraction using Ki-67 antigen expression identifies a subgroup of multiple myeloma patients displaying short survival within the ISS stage I

The current most powerful prognostic model in Multiple Myeloma (MM) combines beta-2 microglobulin (b2m) with albumin, corresponding to the International Staging System (ISS). However, the prognosis of patients within the ISS stage I (high albumin and low b2m) may vary. Ki-67 is a nuclear protein associated with cell proliferation. We retrospectively evaluated the percentage of bone marrow plasma cells expressing Ki-67 antigen (Ki-67 index) in a series of 174 untreated MM patients at diagnosis. Median survival was 51, 41 and 20 months respectively, and median Ki-67 index was 3.0%, 6.1% and 6.5% in ISS stages I, II, and III respectively. Independently of ISS, Ki-67 index > or =4% was highly predictive of adverse prognosis. Ki-67 index correlated with markers of intrinsic malignancy and with markers of tumour burden. Within ISS stage I, median survival was of 31 months (RR of death 2.65) in patients with Ki-67 index > or =4%. Eventually, the combination of Ki-67 with b2m produced an efficient prognostic model, which appeared most effective in our series when compared with b2m and KI-67 with chromosome 13 deletion models. In this series, we demonstrated that a proliferation marker provides clear-cut additional survival prognostic information to b2m into the ISS model.     

Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.     

Clinical significance of sCD105 in angiogenesis and disease activity in multiple myeloma

BackgroundΤhe importance of angiogenesis in malignancies' growth is well recognized. CD105 (Endoglin), a proliferation-associated glycoprotein, is a powerful marker of neovascularization. Elevated amounts of solubleCD105 (sCD105) have been identified in selected solid tumors. The aim of the study was to estimate circulating levels of sCD105 and soluble transforming growth factor-β₁ (sTGF-β₁), in multiple myeloma (MM) patients, to determine their significance in tumor progression and to investigate the correlation between sCD105 and markers of disease activity.MethodsWe studied 50 newly diagnosed MM patients. Twenty-five of them were also investigated in plateauphase. Twenty patients with monoclonal gammopathy of undetermined significance (MGUS) were enrolled in this study. As control group 28 healthy persons were studied. We determined sCD105, sTGF-β₁ and interleukin-6 (IL-6) in the serum, Ki-67 proliferation index (Ki-67 PI) expression and microvascular density(MVD) in bone marrow with immunohistochemistry.ResultsThe mean concentrations of sCD105 and IL-6 were higher in MM and MGUS patients compared to controls, whereas serum levels of sTGF-β₁ were lower in MM patients compared to MGUS patients and controls. sCD105 levels, were significantly different among disease stages, with higher values in advanced stages. It was found that sCD105 correlated with Ki-67 PI, MVD and IL-6.ConclusionsCD105 seems to play an important role in angiogenesis and tumor progression. Circulating levelsof sCD105 could detect patients with more advanced disease and might help in evaluating the response to treatment.     

Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL- 6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF- beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N- BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF- beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.     

Detection of interleukin-6 (IL-6) in human bone marrow myeloma cells by light and electron microscopy

We used an Avidin-Biotin peroxidase complex (ABC) immunoperoxidase technique to evaluate the localization of IL-6 of human bone marrow cells in multiple myeloma (MM). The cellular distribution of IL-6 was determined at light and electron microscopic levels. The author's study indicated that cytoplasmic IL-6 was detected only in the myeloma cells of bone marrow cells. Immunoelectron microscopic (immuno-EM) study showed positive reactivity mainly in the perinuclear space (PNS), well-developed rough endoplasmic reticulum (RER), and Golgi area.     

Clinical significance of interleukin-6 gene expression in the bone marrow of patients with multiple myeloma

The proliferation and differentiation of multiple myeloma (MM) cells has been suggested to be induced by continuous stimulation of interleukin (IL)-6 in the bone marrow (BM) microenvironment. We investigated the production and gene expression of this cytokine in bone marrow mononuclear cells (BMMC) of 31 untreated patients with MM. The IL-6 gene was expressed in 20/31 patients in whom increased IL-6 production in vitro was observed, suggesting that IL-6 gene expression correlates with IL-6 production by BMMC. A significant correlation was found between the presence of bone lesions and IL-6 gene expression; however, the percentage of MM cells infiltrating the BM did not correlate with IL-6 gene expression. This IL-6 gene expression was assigned to adherent cells of the BM microenvironment. Furthermore, serial measurement of IL-6 gene expression along with the clinical course revealed increased expression of IL-6 gene during the progressive phase of MM and decreased expression after remission at the individual level. These results suggest that IL-6 gene expression in BMMC reflects disease severity in MM.