Growth Factor In Multiple Myeloma Research Articles

CD200 Is a Potent New Growth Factor in Multiple Myeloma through IGF-1R Interaction

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the accumulation of clonal plasma cells within the bone marrow.We previously identified that CD200 is expressed in Multiple Myeloma Cells (MMCs) of 78% of newly MMdiagnosed patients and this expression is associated with a bad prognosis. CD200 is a type I immunoglobulin superfamily transmembrane glycoprotein that is expressed by various cell types including B cells and T cells. In tumors cells, interaction of CD200 with its known receptor CD200R, inhibits T-cell mediated immune response and induces immunosuppressive responses. However, the role of CD200 in tumorigenesis is poorly understood. Recently, a study shown that CD200 can induce neuritogenesis and promote neuronal survival of primary neurons through interaction and activation of the fibroblast growth factor receptor.Here, we investigated for the first time the role of CD200 in MM cell biology. First, we identified that CD200 protein expression is present only in 3 out of 9 Human Myeloma Cell lines (HMCLs). Then, we confirmed that CD200 protein is strongly expressed in MMCs of 73% of the 35 previously untreated MM patients and that CD200 is never expressed in the normal plasma cell counterparts.CD200 overexpression using lentiviral vector significantly increased HMCLs cell growth in a serum free culture medium (SYN-H) compared to GFP overexpression (fold increasing: 1.6 and 1.4 at day 4; 2.5 and 2.1 at day 7 for LP1 and XG1 respectively). Furthermore, CD200 overexpression results in significant increasing of the number of colonies for HMCLs CD200positive compared to HMCLs CD200negative in clonogenic assays (4.3 fold and 13.1 fold for LP1 and XG1 respectively, p<0.001). We confirmed also the growth advantage linked to CD200 overexpression in coculture model. These effects are significantly inhibited by a monoclonal antibody (MoAb) against CD200 compared to treatment with a no-relevant anti-IgG1. We also confirmed the significant inhibition of proliferation with the anti-CD200 MoAb using clonogenic assays. The anti-CD200 MoAb has no effect on the GFP transduced HMCLs.To investigate the implication of CD200R in CD200-induced MM cell growth, inducible CD200R depletion was analyzed in CD200positive and CD200negative, using lentiviral vectors. After doxycycline addition, the shCD200R reduced CD200R expression by 95-100% but had no significant effects on the proliferation advantage mediated by CD200 demonstrating that the growth advantage supported by CD200 overexpression is independent of CD200R.Thus, to investigate the possibility of another receptor activated by CD200 interaction, we added different essential MM growth factors to the serum free medium. Addition of fetal calf serum, IGF1 or insulin in SYN-H medium abrogated the growth advantage provided by CD200 overexpression in HMCLs. These data suggest that CD200 could act as a growth factor through interaction and activation of IGF1R. In addition, overexpression of CD200 in a IGF-1R negative XG12 HMCL did not stimulate its cell growth or clonogenic activity. Furthermore, anti-IGF-1R monoclonal antibody completely abrogated the CD200-dependent growth activity at day 4 and at day 7 in LP1CD200positive cell line compared to non-relevant antibody. The anti-IGF-1R MoAb has no effect on control LP1GFPpositive cell line cultured in serum free culture medium.In order to identify a direct interaction between CD200 and IGF-1R in serum free culture conditions, IGF-1R immunoprecipitation in denaturing conditions was performed at day 4 using LP1GFP and LP1CD200 cell lines. CD200 and IGF-1R interaction was identified by mass spectrometry and western-blotting.According to these data, MM patients with CD200high and IGF1Rhigh gene expression have a poor prognosis (OS) compared to patient with only CD200high or IGF1Rhigh expression (P=0.03, 52.7 vs 61.9 months in HM series; and P=0.02, 58.9 vs 68.1 months in TT2 series) and compared to patients with CD200low and IGF1Rlow gene expression (P=0.002, 52.7 vs 71.6 months in HM series; and P=0.01, 58.9 vs 70.2 months in TT2 series). Same results are observed for EFS.Altogether, these data demonstrated a new role of CD200 as a MM growth factor through interaction with IGF1R. Our results suggest that CD200 and IGF1R inhibition by monoclonal antibodies could represent a promising strategy in MM. DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:Sanofi: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; EngMab: Research Funding.

Targeting Insulin-like Growth Factor in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant Cells

Introduction: Multiple myeloma (MM) is one of the hematological malignancy that affects the plasma cells in bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs. However, most patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Insulin-like growth factor (IGF) cause intracellular signaling that ultimately results in cellular growth and proliferation. Because IGF signaling pathways have crucial functions in hematological malignancies and solid tumors, IGF pathways might regulate myeloma cell survival.Materials and Methods: In this study, we investigated whether IGF1R inhibitor could suppress myeloma cells. We also investigated whether IGF1 was involved in proteasome sensitivity against myeloma cells and cytokines were induced by IGF1 in human umbilical vein endothelial cells (HUVEC).Results: We first investigated the IGF signaling pathways by microarray gene expression data from the online Gene Expression Omnibus (GEO). IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Gene expression of IGF1, IGF1R, IGFBP2, IGFBP5, IGFBP7 and IGF2 are involved in myeloma and normal control samples from the public microarray datasets of GSE39754. We next investigated the proteasome inhibitor, bortezomib or carfilzomib activity. Proteasome inhibitors exhibit cell growth inhibition against myeloma cells. However, in the presence of IGF1, proteasome inhibitors activity was reduced. Linsitinib is an orally bioavailable small molecule inhibitor of the IGF1R. We investigated whether linsitinib could inhibit the proliferation of myeloma cells. We found linsitinib treatment inhibited the growth of myeloma cells in a dose dependent manner. Activity of Caspase 3/7 and cytotoxicity were also increased. Combined treatment of myeloma cells with carfilzomib and linsitinib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We examined the intracellular signaling. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after carfilzomib and linsitinib treatment. We next blocked IGF1R function by small interfering RNA (siRNA). We found drug sensitivity of the cells to the carfilzomib was increased compared to mock-transfected cells. Caspase 3/7 activity and cytotoxicity were increased after carfilzomib treatment in siRNA transfected cells.We next investigated the in vitro effects of IGF1 on cultured HUVEC. Because the entire bone marrow cavity is hypoxic condition, we investigated the IGF signaling pathways in the hypoxic condition by microarray gene expression data from GEO. Gene expression of IGF1R and IGF2 are increased in the hypoxia from the public microarray datasets of GSE36837. Gene amplification was confirmed by RT-PCR analysis. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IGF1 stimulation in the time dependent manner. We also found that gene expression of monocyte chemotactic protein-1 (MCP-1) was enhanced by IGF1 stimulation.Conclusion: The IGF1 signaling pathway is involved in proteasome inhibitor sensitivity and plays a key role in chemokine production of the HUVEC. Our data also suggested that administration of IGF1R inhibitor, linsitinib, might be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. DisclosuresOhyashiki:MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Jansen Pharma KK,: Research Funding; Chugai KK,: Honoraria, Research Funding; Nihon-Seiyaku,: Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Eizai,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Asahikase: Research Funding; Asteras KK,: Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding.

Carbamazepine and Hematological Malignancies

Received/Gelis tarihi : May 1, 2013 Accepted/Kabul tarihi : June 10, 2013 To the Editor, The letter entitled “Carbamazepine and Multiple Myeloma: Possible Interaction”, written by Gunaldi et al. [1] and published in one of the recent issues of your journal, was quite interesting. We would like to emphasize some points about that letter. In the reported case, the patient’s serum globulin level was 5.44 g/dL, and serum protein electrophoresis (Figure 1 of the cited letter) showed that the M-peak was closest to the beta-fraction. Thus, it becomes important to know the level of light chains and IgA as heavy chains. The serum protein electrophoresis should also be evaluated for biclonal gammopathy due to its pattern shown in Figure 1. Interleukin-6 (IL-6) is the main growth factor for multiple myeloma (MM) and studies of epileptic patients showed elevated levels of IL-6 after carbamazepine therapy [2]. We have no information about the level of IL-6 in the case reported by Gunaldi et al. The specific abnormalities such as t(4;14), t(14;16), and deletion (del) 17p detected by fluorescence in situ hybridization (FISH) analysis were reported as high-risk MM [3]. Tricot et al. [4] reported that partial or complete deletions of chromosome 13 were associated with poor prognosis in MM. Chromosome 13 deletions detected only by FISH independently in the absence of other abnormalities do not carry significantly higher risk, whereas t(11;14) does not predict superior outcome [5]. It is interesting to see cytogenetic abnormalities del17p-t(4;14)-del13 and t(11;14) in same patient. However, the consensus is that the data are not yet adequate to suggest routine use of these FISH markers to predict prognosis [5]. The number of people who had suffered from side effects due to carbamazepine was reported as 14,705. Ten of them (0.07%) had MM [6]. Drug-related (although rarely from carbamazepine) hypogammaglobulinemia is a well-known condition. 9-Acridine carboxaldehyde is one of the carbamazepine metabolites generating activated neutrophils, and presumably monocytes. This metabolite increases lymphocyte proliferations at lower concentrations and decreases them at higher concentrations. The relationship between hypogammaglobulinemia and carbamazepine is thus clearer [7]. There have also been reports of agranulocytosis, leucopenia, pure erythrocyte aplasia, and thrombocytopenia after carbamazepine [8]. Additionally, phenytoin is considered a possible carcinogenic for humans and was reported as a carcinogenic drug in animals [9]. A diagnosis of epilepsy in the same year as a cancer diagnosis carried an increased risk for leukemia, pancreatic cancer, non-Hodgkin’s lymphoma, acute myeloid leukemia, and Hodgkin’s disease, but not for chronic lymphatic leukemia or MM. The issue of cancer incidence in people with epilepsy remains an open question [9,10]. In conclusion, the answer is still unknown as to whether the drugs for epilepsy or epilepsy itself causes MM.

Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma

BackgroundThe ELR+ CXC chemokines are important mediators of tumorigenesis, related to their angiogenic properties. Angiogenesis appears to be a prominent feature in the progression of multiple myeloma (MM). CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine molecules separated by a single amino acid. The angiogenic potential of this group is determined by the presence of three amino acid residues (Glu-Leu-Arg: the ELR motif) preceding the first cysteine amino acid, in the NH2 terminus. AimsThe purpose of this study was to determine serum concentrations of angiogenesis-related chemokines ELR+ motif, such as interleukin-8 (IL-8), epithelial neutrophil activating protein-78 (ENA-78) and growth-related gene alpha (GRO-α), as well the bone marrow microvascular density (MVD) in patients with MM at diagnosis and after treatment, in plateau phase. We also evaluated the relationship among them with other known growth factors involved in angiogenesis. MethodsSerum levels of the ELR+ CXC chemokines: IL-8, ENA-78 and GRO-α as well as of the angiogenic factors: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were determined in 63 newly diagnosed MM patients, in 30 in plateau phase and in 20 healthy controls. Serum measurements of them were performed with commercially available kits for ELISA. Bone marrow biopsies were performed before and after treatment, in plateau phase, in order to determine MVD by staining vessels with anti-CD31. ResultsSerum concentrations of IL-8, ENA-78, GRO-α and TNF-α were significantly higher in the group of MM patients (44.5±25.3, 765±572.1, 186.5±129.1 and 4.2±2.8pg/ml, respectively) in comparison to control group (27.3±6.4, 335.1±268.6, 112.5±76.1 and 1.3±0.8pg/ml) (p<0.02 for GRO-α, p<0.001 for other cases). We also found that untreated patients had higher levels of IL-8, ENA-78, GRO-α than post treatment patients, but statistical significant difference was found only for IL-8 (48.36±30.93pg/ml vs. 35.05±19.77pg/ml, p<0.001). Furthermore IL-8, GRO-α, TNF-α, HGF and VEGF were significantly higher with increasing disease stage (p<0.001 in all cases). ENA-78 serum levels were higher in stage III than in stage I and II, but without statistical significance. Additionally we correlated each proinflammatory cytokine with well known angiogenic factors such as HGF, VEGF and TNF-α. A positive correlation was found between serum HGF and IL-8 and GRO-α (r=0.316 p<0.01, r=0.297 p<0.02, respectively). Similarly serum VEGF correlated with ENA-78 and GRO-α (r=0.323 p<0.01, r=0.469 p<0.001, respectively). In the pretreatment group of patients a positive correlation between bone marrow MVD and serum levels of GRO-α was found (r=0.304 p<0.01). There was a difference in survival times between patients with higher than median versus low IL-8, ENA-78 and GRO-α levels, but the differences could not reach statistical significance in either case. ConclusionsThese findings support the hypothesis that ELR+ motif CXC chemokines, such as IL-8, ENA-78 and GRO-α correlate with angiogenic growth factors and may play a role in the progression of MM. Further studies are needed to determine their prognostic and predictive significance.

Stem Cell Transplant without Growth Factor In Multiple Myeloma: Engraftment Kinetics, Bacteremia, and Hospitalization.

AbstractAbstract 3469 Introduction:Stem cell transplantation is important in the management of multiple myeloma. In the United States, the standard of care is administration of growth factors to accelerate neutrophil recovery after stem cell transplant. The need for growth factors after transplant has not been investigated recently. Patients:We analyzed a cohort of 166 patients at our institution who underwent autologous transplant for multiple myeloma without receiving growth factors after transplant and compared them with 498 patients who received standard filgrastim beginning on posttransplant day 5. TABLE Results:A neutrophil count of 500/μL was achieved in a median of 12.5 days in patients receiving growth factor, compared with 13.5 days in those not receiving growth factor (P<.001) Fig 1. Platelet engraftment was identical (median, 14.5 days; P=.12) in both groups, despite a lower median number of CD34+ cells infused in patients who did not receive growth factors. Incidence of nonstaphylococcal bacteremia was identical in both groups. The median hospital stay was 3.5 days shorter in the group not receiving growth factor. Bacteremia impacted platelet but not neutrophil engraftment. Figure 2 Conclusion:It is feasible and reasonable to perform autologous stem cell transplant for multiple myeloma without administering growth factors. Estimated savings would be $4,600 for each transplanted patient. Growth factor administration is not required for a safe outcome and decreases the number and severity of some of the fluid-related complications after transplant such as acute respiratory distress syndrome, allergic reactions, alveolar hemorrhage, and rarely splenic rupture.TablePatient CharacteristicsPatient GroupCharacteristicFilgrastim (n=498)No Filgrastim (n=166)P ValueMen290 (58)102 (61).52Age, y59.8 (53.0–65.4)61 (53.9–66.8).25Serum M spike, g/dL0.8 (0.1–1.8)0.6 (0.1–1.8).22Urine total protein, g/24 h0.10 (0.06–0.33)0.09 (0.05–0.28).67b2M, mcg/mL2.56 (1.97–3.71)2.71 (2.10–3.66).13LDH, units/L187 (158–229)184 (152–214).35Myeloma bone disease422 (85)146 (88).37Marrow PCs, %8 (2–21)8 (2–23).60Radiotherapy137 (28)48 (29).76Abnormal metaphase cytogenetics98 (20) (n=494)27 (16) (n=165).36Albumin, g/dL3.7 (3.3–4.0)3.3 (3.1–3.4)<.001Creatinine, mg/dL1.0 (0.9–1.2)0.9 (0.8–1.1).002ISSStage I222 (46) (n=481)21 (13) (n=165)<.001Stage II200 (42)125 (76)<.001Stage III59 (12)19 (12)NACD34+ cells infused, ×106 cells/kg4.33 (3.78–5.51)4.19 (3.50–5.03).01All-cause mortality before day +10010 (2)3 (2)NA [Display omitted] [Display omitted] Disclosures:Gertz:Celgene: Honoraria; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lacy:Celgene: Research Funding. Dispenzieri:Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

SCT without growth factor in multiple myeloma: engraftment kinetics, bacteremia and hospitalization

SCT is important in the management of multiple myeloma. In the United States, the standard of care is administration of growth factors to accelerate neutrophil recovery after SCT. The need for growth factors after transplant has not been investigated recently. We analyzed a cohort of 166 patients at our institution who underwent autologous transplant for multiple myeloma without receiving growth factors after transplant and compared them with 498 patients who received standard filgrastim beginning on posttransplant day 5. A neutrophil count of 500/μL was achieved in a median of 12.5 days in patients receiving growth factor, compared with 13.5 days in those not receiving growth factor (P<0.001). Platelet engraftment was identical (median, 14.5 days; P=0.12) in both groups, despite a lower median number of CD34+ cells infused in patients who did not receive growth factors. Incidence of nonstaphylococcal bacteremia was identical in both groups. The median hospital stay was 3.5 days shorter in the group not receiving growth factor. It is feasible and reasonable to perform autologous SCT for multiple myeloma without administering growth factors.

Growth factors in multiple myeloma: a comprehensive analysis of their expression in tumor cells and bone marrow environment using Affymetrix microarrays

BackgroundMultiple myeloma (MM) is characterized by a strong dependence of the tumor cells on their microenvironment, which produces growth factors supporting survival and proliferation of myeloma cells (MMC). In the past few years, many myeloma growth factors (MGF) have been described in the literature. However, their relative importance and the nature of the cells producing MGF remain unidentified for many of them.MethodsWe have analysed the expression of 51 MGF and 36 MGF receptors (MGFR) using Affymetrix microarrays throughout normal plasma cell differentiation, in MMC and in cells from the bone marrow (BM) microenvironment (CD14, CD3, polymorphonuclear neutrophils, stromal cells and osteoclasts).Results4/51 MGF and 9/36 MGF-receptors genes were significantly overexpressed in plasmablasts (PPC) and BM plasma cell (BMPC) compared to B cells whereas 11 MGF and 11 MGFR genes were overexpressed in BMPC compared to PPC. 3 MGF genes (AREG, NRG3, Wnt5A) and none of the receptors were significantly overexpressed in MMC versus BMPC. Furthermore, 3/51 MGF genes were overexpressed in MMC compared to the the BM microenvironment whereas 22/51 MGF genes were overexpressed in one environment subpopulation compared to MMC.ConclusionsTwo major messages arise from this analysis 1) The majority of MGF genes is expressed by the bone marrow environment. 2) Several MGF and their receptors are overexpressed throughout normal plasma cell differentiation. This study provides an extensive and comparative analysis of MGF expression in plasma cell differentiation and in MM and gives new insights in the understanding of intercellular communication signals in MM.

Coexistence of primary AL amyloidosis and POEMS syndrome: Efficacy of melphalan‐dexamethasone and role of biochemical markers in monitoring the diseases course

American Journal of HematologyVolume 85, Issue 2 p. 131-132 LetterFree Access Coexistence of primary AL amyloidosis and POEMS syndrome: Efficacy of melphalan-dexamethasone and role of biochemical markers in monitoring the diseases course† Fausto Adami, Corresponding Author Fausto Adami [email protected] Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalyDepartment of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Via Giustiniani 2, Padova 35128, ItalySearch for more papers by this authorChiara Briani, Chiara Briani Department of Neurosciences, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGianni Binotto, Gianni Binotto Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorSara Altinier, Sara Altinier Department of Laboratory Medicine, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGiuseppe Tarantini, Giuseppe Tarantini Department of Cardiac, Thoracic and Vascular Sciences, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGianpietro Semenzato, Gianpietro Semenzato Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalySearch for more papers by this author Fausto Adami, Corresponding Author Fausto Adami [email protected] Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalyDepartment of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Via Giustiniani 2, Padova 35128, ItalySearch for more papers by this authorChiara Briani, Chiara Briani Department of Neurosciences, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGianni Binotto, Gianni Binotto Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorSara Altinier, Sara Altinier Department of Laboratory Medicine, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGiuseppe Tarantini, Giuseppe Tarantini Department of Cardiac, Thoracic and Vascular Sciences, Padova University School of Medicine, Padova, ItalySearch for more papers by this authorGianpietro Semenzato, Gianpietro Semenzato Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, Padova University School of Medicine, Padova, ItalySearch for more papers by this author First published: 29 October 2009 https://doi.org/10.1002/ajh.21581Citations: 3 † Conflict of interest: Nothing to report. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Kuwabara S. New strategy of treatment for POEMS syndrome: Autologous peripheral blood stem cell transplantation and thalidomide therapy. Brain Nerve 2008; 60: 627– 633. 2 Dispenzieri A. POEMS syndrome. Blood Rev 2007; 21: 285– 299. 3 Kuwabara S,Dispenzieri A,Arimura K, et al. Treatment for POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) syndrome. Cochrane Database Syst Rev 2008; 4: CD006828. 4 Dispenzieri A,Kyle R,Merlini G, et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009; 23: 215– 224. 5 Iwasaki T,Hamano T,Ogata A, et al. Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma. Br J Hematol 2002; 116: 796– 802. 6 Soubrier M,Dubost JJ,Serre AF, et al. Growth factors in POEMS syndrome: Evidence for a marked increase in circulating vascular endothelial growth factor. Arthritis Rheum 1997; 40: 786– 787. 7 Scarlato M,Previtali SC,Carpo M, et al. Polyneuropathy in POEMS syndrome: Role of angiogenic factors in the pathogenesis. Brain 2005; 128: 1911– 1920. 8 Nobile-Orazio E,Terenghi F,Giannotta C, et al. Serum VEGF levels in POEMS syndrome and in immune-mediated neuropathies. Neurology 2009; 72: 1024– 1026. 9 Patier JL,Pinilla A,Rodriguez-Posada A, et al. Primary amyloidosis associated with osteosclerotic myeloma. Rev Clin Esp 2003; 203: 162– 163. 10 Briani C,Scarlato M,Pavan A, et al. Polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes: Not always a POEMS syndrome. Hematologica 2006; 91: ECR10. 11 Watanabe O,Maruyama I,Arimura K, et al. Overproduction of vascular endothelial growth factor/vascular permeability factor is causative in Crow-Fukase (POEMS) syndrome. Muscle Nerve 1998; 21: 1390– 1397. 12 Merlini G,Stone MJ. Dangerous small B-cell clones. Blood 2006; 108: 2520– 2530. 13 Palladini G,Perfetti V,Obici L, et al. Association of melphalan and high-dose dexamethasone is effective and well tolerated in patients with AL (primary) amyloidosis who are ineligible for stem cell transplantation. Blood 2004; 103: 2936– 2938. Citing Literature Volume85, Issue2February 2010Pages 131-132 ReferencesRelatedInformation

Interleukin-6-independent expression of glucocorticoid receptor is upregulated by triptolide in multiple myeloma

Glucocorticoids are widely used chemotherapeutic agents for multiple myeloma. Drug resistance to steroid therapies is associated with the downregulation or loss of glucocorticoid receptor expression in malignant plasma cells. In this study, we examined the constitutive expression of glucocorticoid receptor in dexamethasone-sensitive and dexamethasone-resistant multiple myeloma cell lines. We found that triptolide increased the amount of the phosphorylated glucocorticoid receptor and enhanced the growth inhibitory effect of dexamethasone. Notably, these effects could not be blocked by interleukin-6, one of the most important growth factors in multiple myeloma.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

The fibroblast growth factors in multiple myeloma

Fibroblast growth factor receptor 3 (FGFR3) is a membrane-anchored receptor tyrosine kinase that transmits intercellular signals mediated by a family of fibroblast growth factors (FGFs). In multiple myeloma (MM), 15% of patients markedly upregulate FGFR3 as a consequence of a t(4;14)(p16.3;q32) translocation. Ectopic expression of FGFR3 enhances MM cell proliferation and survival, demonstrating the oncogenic potential of FGFR3.

Insuline-Like Growth Factor 1 Is over Expressed in Multiple Myeloma Plasma Cells and Regulates the Expression of the Insuline-Like Growth Factor 1 Receptor.

Insulin-like growth factors 1 (IGF1) plays an important role in regulating cell proliferation, differentiation, apoptosis, and transformation. IGF1 acts by binding the type 1 IGF receptor (IGF-1R), thus activating its tyrosine kinase activity. Recent studies have shown that IGF1 is an important survival and growth factor in multiple myeloma (MM).In this study we evaluated the expression of IGF1 and IGF1-R in a series of 49 newly diagnosed MM patients (pts) primarily treated with thalidomide and dexamethasone. The aim of the study was to correlate the expression of these genes with presenting features of MM pts and to evaluate their relationship with response to therapy.We isolated the CD138+ cell fraction from bone marrow (BM) samples; IGF1 and IGF1-R expression was evaluated by Real-time RT PCR in both CD138+ and CD138- cells, considering a pool of donors as calibrator (expression value=1). We found that neoplastic CD138+ and CD138- cells expressed widely high levels of IGF1 (median 145.01 and 3.16, range 0.13–1089.92 and 0.02–103.25, respectively), and low levels of IGF1-R (median 0.76 and 2.27, range 0.04–9.38 and 0.21–13.55, respectively). As expected, expression data for both genes, in both cell populations resulted very scattered; in fact no differences could be highlighted in the expression of IGF1 and IGF1-R genes, with respect neither to presenting clinical features, nor to the presence of two of the most frequent MM chromosomal alterations (t(4;14) and del(13)). According to response to induction therapy, pts were divided in two subgroups, including 35 responsive and 14 non responsive pts, respectively. Again, overall no relationship was pointed out between the expression of IGF1 and IGF1-R and response to therapy.IGF1 is known to regulate IGF1-R at transcriptional level, down-regulating its expression; this mechanism may be autocrine or paracrine. We thus hypothesized that the unexpectedly low IGF1-R expression levels might be the consequence of a negative-feedback regulation mechanism, induced by an IGF1autocrine effect. Nevertheless, we were not able to highlight a significant inverse correlation between the expression values of IGF and IGF1-R in any of the cell fraction analyzed. On the contrary, among responsive pts, a highly significant inverse correlation was highlighted between IGF1 expressed by the CD138+ and IGF1-R expressed by the CD138− cells (p= 0.001), thus suggesting a possible paracrine effect of IGF1 produced by plasma cells exerted on CD138− cells; interestingly, the same effect could not be highlighted in pts non responding to induction therapy. In conclusion, our preliminary data confirmed the possible involvement of IGF1/IGF1R pathway in MM pathogenesis; we suggested the existence of a paracrine effect of IGF1 produced by CD138+ cells, thus emphasizing the importance of the dialog between plasma cells and microenvironment in MM. Moreover, as this mechanism seemed to act only in responding pts, the ability to efficiently regulate IGF1-R expression may have an important prognostic value. The study of post transduction modifications of the IGF1-R will thus be needed, in order to get more insight into the relationship between the IGF and IGF1-R expressions and the receptor activation.

Bone marrow microvascular density and angiogenic growth factors in multiple myeloma

There is evidence that angiogenesis plays an important role in the progression of multiple myeloma (MM). Hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF-alpha) are cytokines that potently stimulate angiogenesis. We evaluated the microvascular density (MVD) of bone marrow biopsies (after immunostaining with anti-CD34 antibodies) and serum levels of HGF and TNF-alpha in 43 patients with newly diagnosed MM. Twenty-four of these patients reached a plateau phase after treatment and were reevaluated for MVD, HGF and TNF-alpha. MVD values and serum levels of HGF and TNF-alpha were elevated in newly diagnosed MM patients in comparison with healthy controls. Pre-treatment MVD, HGF and TNF-alpha increased with advancing stage of MM disease. In patients reaching the plateau phase, a significant reduction in MVD, HGF and TNF-alpha levels occurred. A positive correlation was noted between pre-treatment MVD and serum levels of TNF-alpha and lactic dehydrogenase but not with HGF. However, HGF strongly correlated with beta2-microglobulin (beta2M), TNF-alpha and lactate dehydrogenase (LDH). We conclude that angiogenesis in MM, as expressed by the bone marrow MVD and the serum levels of angiogenic molecules such as HGF and TNF-alpha, increases with advancing clinical stage and decreases after effective chemotherapy.

A review of the cytokine network in multiple myeloma: diagnostic, prognostic, and therapeutic implications.

Because many studies have focused on growth factors in multiple myeloma, the study of the cytokine network appears to be useful for this purpose. Interleukin-6 (IL-6) and IL-2 with their soluble receptors (IL-3, IL-4, IL-10, and IL-11) have been examined. Plasma cells may produce IL-6 by an autocrine mechanism whereas a paracrine mechanism is believed to be involved in the production of IL-6 by bone marrow stromal cells through an interaction between adhesion molecules present on myeloma plasma cells and their respective receptors that are present on bone marrow stromal cells. In addition, control over production of IL-6 may be exerted by other ILs such as IL-1beta and IL-10. Among target cells, the growth of normal and myeloma plasma cells is supported by IL-6, which also induces the differentiation of myeloma plasmablastic cells into mature plasma cells. This last action also is shared by IL-3, IL-4, and, most likely, IL-8. Evaluation of the serum level of IL-6, C reactive protein, soluble IL-6 receptor (sIL-6R), and soluble IL-2 receptor (sIL-2R), together with the activity exerted by IL-3 and IL-4 on some cellular subsets, may constitute an additional element in the differential diagnosis of borderline cases. However, the concomitant evaluation of all immunologic parameters could be more useful than the value of a single IL. Serum levels of IL-6, sIL-6R, sIL-2R, and the expression of membrane-bound IL-2 receptors, both on bone marrow plasma cells and on peripheral blood mononuclear cells, are correlated with disease activity and disease stage. In addition, IL-6 and sIL-6R serum levels are believed to be correlated with the duration of disease-free survival because a high serum level at the time of diagnosis is believed to be correlated with a short duration of survival. However, some laboratory parameters may express the same prognostic value as high beta(2) microglobulin and lactate dehydrogenase (LDH) serum levels together with a high plasma cell labeling index are correlated with disease activity. Furthermore, if the evaluation is performed at the time of diagnosis, high values of these parameters are correlated with a short disease-free survival. A correlation between laboratory parameters and the serum level of several cytokines was demonstrated. Hence, the real advantage of the prognostic evaluation of cytokines is reserved for patients who do not exhibit uniform results with regard to beta(2) microglobulin and LDH serum levels, or, better, for borderline cases. With regard to the differential diagnosis, all immunologic parameters should be evaluated concomitantly rather than separately to confer a real prognostic value to results. Furthermore, a particular relation was found between a high sIL-6R serum level and a poor response to chemotherapy, therefore suggesting the possibility of identifying in advance a subset of patients with a high risk of treatment failure, as has already been demonstrated in other hematologic malignancies.Finally, the majority of studies indicate that interferons are used mainly in the immunotherapy for multiple myeloma, whereas many clinical trials should still be required for the evaluation of the effectiveness of anti-I-L6 antibodies or antiidiotypic vaccines in reference to the eligible patients for these particular therapies.

Relationship between circulating serum soluble interleukin-6 receptor and the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in multiple myeloma.

Angiogenesis plays an important role in multiple myeloma (MM) progression. Various mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) have been implicated in the angiogenic process of various malignancies. Interleukin-6 (IL-6) is a growth factor of myeloma cells and its signaling is mediated via a cell surface receptor complex (IL-6r). IL-6 and tumor necrosis factor-alpha (TNF-alpha) are involved in the secretion of VEGF by IL-6r expressing myeloma cells. In this study, serum FGF-2, VEGF, IL-6r, and TNF-alpha were measured in 46 untreated MM patients and were studied in relation to disease stage (by Salmon-Durie criteria) and severity [assessed by serum beta(2)-microglobulin (beta(2)M), C-reactive protein (CRP), alpha(1)-antitrypsin (alpha(1)AT), and lactic dehydrogenase (LDH) levels]. The results showed that FGF-2, VEGF, IL-6r, and TNF-alpha were significantly elevated in MM patients in comparison to controls ( p<0.008) and were significantly higher in stage III disease in comparison to stages I and II ( p<0.03). The mean concentrations of IL-6r were 877+/-374, 1220+/-308, 1431+/-878, and 453+/-180 pg/ml for stages I, II, and III and controls, respectively. Levels of beta(2)M, alpha(1)AT, CRP, and LDH were all significantly higher in MM patients than controls and increased with advancing stage of disease. There were positive correlations of both VEGF and FGF-2 with IL-6r, TNF-alpha, beta(2)M, alpha(1)AT, CRP, and LDH. We conclude that IL-6r and TNF-alpha increase in parallel to VEGF and FGF-2 with increasing stage of MM disease. These molecules correlate with biochemical markers of disease activity and may play a role in the progression of multiple myeloma.

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.

Effects of IL-6 Variants in Multiple Myeloma: Growth Inhibition and Induction of Apoptosis in Primary Cells

Interleukin-6 (IL-6) plays a pathogenetic role in B-cell malignancies and is a growth factor for multiple myeloma (MM) cells. Elevated serum IL-6 levels and a higher proliferative activity of bone marrow plasma cells are poor prognostic factors in MM patients. In addition to clinical trials with anti-IL-6 monoclonal antibodies, an alternative therapeutic approach based on the use of IL-6 receptor (R) super-antagonists (Sants) has been proposed. Sants are variants of the native cytokine characterized by a wild type affinity for the ligand-specific receptor chain IL-6R alpha and by a reduced ability to bind and/or dimerize the signaling chain gp-130. We report the in vitro effects of four different Sants on cell kinetic modulation and induction of apoptosis of primary cells from MM patients. Ten MM samples were cultured in the presence of four different Sants and heterogeneous effects in terms of reduction of proliferation and induction of apoptosis could be observed. A decrease of the S phase cells (≥ 25%) coupled with the induction of apoptosis was obtained in 4/10 samples: three of these samples had a diploid DNA stem line and an inferior initial percentage of S phase cells. Serum IL-6 concentrations did not correlate with the anti-proliferative activities of the Sants. Cell growth inhibition was observed especially in samples with soluble IL-6R serum concentrations >200 ng/ml. We conclude that Sants can exert antiproliferative effects on selected MM samples. Such effects may depend on the availability of large amounts of soluble IL-6R. Further studies should aim at defining the conditions necessary for optimal antiproliferative activity.

Dexamethasone plus retinoids decrease IL-6/IL-6 receptor and induce apoptosis in myeloma cells.

Interleukin 6 (IL-6) is the most important known growth factor for multiple myeloma, and IL-6 signalling pathways are potential targets for therapy. We hypothesized that interfering with the IL-6 signalling pathway at more than one level would be more effective than a single block in inhibiting proliferation of myeloma cells. Accumulating data support the concept that glucocorticoids down-regulate IL-6, whereas retinoic acid derivatives (RA) down-regulate IL-6R in myeloma. We found that all-trans RA (ATRA), 13-cis-RA and 9-cis-RA each similarly inhibited growth of RPMI 8226 myeloma cells and that addition of dexamethasone (DEX) added to RA growth inhibition. The major effects of retinoids were to reduce the proliferative fraction and induce apoptosis whereas DEX increased the apoptotic fraction. When combined, apoptosis was enhanced. Effects of RA + DEX were also least able to be overcome by exogenous IL-6. RA decreased IL-6R levels and addition of DEX to RA delayed recovery of IL-6R levels compared with RA alone. Since RPMI 8226 cells have undetectable IL-6, we investigated U266B1 cells and found that RA and DEX decreased both IL-6 secretion and IL-6 RNA levels. Mechanistically, IL-6R down-regulation by RA was enhanced by DEX, whereas IL-6 protein and RNA levels were reduced by DEX and by RA. In summary, combinations of RA + DEX were not only more effective in inhibiting myeloma cells growth by the dual mechanisms of decreasing proliferative fraction and increasing apoptotic fraction, but were also less able to be overcome by IL-6.

Interleukin-6 Overcomes p21WAF1 Upregulation and G1 Growth Arrest Induced by Dexamethasone and Interferon-γ in Multiple Myeloma Cells

Interleukin-6 (IL-6) is a growth factor for multiple myeloma (MM) cells and can inhibit MM cell apoptosis. Our recent studies show that IL-6 facilitates MM cell growth via phosphorylation of retinoblastoma protein (pRB); however, the effects of IL-6 on those cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDIs) that are known to regulate phosphorylation of pRB have not been defined in MM cells. In the present report, we cultured MM cell lines and patient cells with IL-6 and/or dexamethasone (Dex) and characterized changes in cell cycle; expression and association of cyclins, CDKs, and CDIs; and phosphorylation of pRB. Dex induced G1 growth arrest in MM cells, whereas IL-6 facilitated G1 to S phase transition; moreover, the effect of Dex was blocked by IL-6. p21WAF1 (p21) protein was constitutively expressed in the majority of MM cells independent of the status of p53. Its expression was upregulated by Dex and downregulated by IL-6; again, IL-6 inhibited the increase in p21 triggered by Dex. These alterations in p21 expression in MM cells were associated with changes in p21 binding to CDK2, CDK4, and CDK6; CDK2, CDK4, and CDK6 kinase activities; and phosphorylation of pRB. In contrast, expression of G1 cell cycle regulatory proteins, including p27KIP1, cyclin D2, and cyclin E, was not altered in MM cells cultured with Dex and/or IL-6. Finally, interferon-γ (IFN-γ) also induced G1 growth arrest and upregulated p21 protein expression; as with Dex, affects of IFN-γ were inhibited by IL-6. Our results therefore show that changes in cell cycle distribution in MM cells triggered by Dex, IL-6, and IFN-γ correlate with changes in p21 protein expression and implicate p21 in the coupling of Dex-, IL-6–, and IFN-γ–related signals to G1 cell cycle regulation in MM cells.

Interleukin-6 Overcomes p21WAF1 Upregulation and G1 Growth Arrest Induced by Dexamethasone and Interferon-γ in Multiple Myeloma Cells

AbstractInterleukin-6 (IL-6) is a growth factor for multiple myeloma (MM) cells and can inhibit MM cell apoptosis. Our recent studies show that IL-6 facilitates MM cell growth via phosphorylation of retinoblastoma protein (pRB); however, the effects of IL-6 on those cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDIs) that are known to regulate phosphorylation of pRB have not been defined in MM cells. In the present report, we cultured MM cell lines and patient cells with IL-6 and/or dexamethasone (Dex) and characterized changes in cell cycle; expression and association of cyclins, CDKs, and CDIs; and phosphorylation of pRB. Dex induced G1 growth arrest in MM cells, whereas IL-6 facilitated G1 to S phase transition; moreover, the effect of Dex was blocked by IL-6. p21WAF1 (p21) protein was constitutively expressed in the majority of MM cells independent of the status of p53. Its expression was upregulated by Dex and downregulated by IL-6; again, IL-6 inhibited the increase in p21 triggered by Dex. These alterations in p21 expression in MM cells were associated with changes in p21 binding to CDK2, CDK4, and CDK6; CDK2, CDK4, and CDK6 kinase activities; and phosphorylation of pRB. In contrast, expression of G1 cell cycle regulatory proteins, including p27KIP1, cyclin D2, and cyclin E, was not altered in MM cells cultured with Dex and/or IL-6. Finally, interferon-γ (IFN-γ) also induced G1 growth arrest and upregulated p21 protein expression; as with Dex, affects of IFN-γ were inhibited by IL-6. Our results therefore show that changes in cell cycle distribution in MM cells triggered by Dex, IL-6, and IFN-γ correlate with changes in p21 protein expression and implicate p21 in the coupling of Dex-, IL-6–, and IFN-γ–related signals to G1 cell cycle regulation in MM cells.