- Email: [email protected]
B587 A Novel Genetic Polymorphism in the DNA Polymerase Kappa Gene May Play a Role in Multiple Myeloma
Basic Biology and Preclinical Studies have been described to play roles in solid tumors and hematologic malignancies; however the role of miRNAs in MM has not been yet fully described. Methods: We performed miRNA-expression-profiling of bone marrow-derived CD138 MM cells isolated from patients included in RAD001 clinical trial, compared to their normal cellular counterparts and validated data by qRT-PCR. MM cell lines (MM.1S/ RPMI8226/U266) were also studied. In vitro and in vivo functional studies were performed on miRNA-15a- and -16-1-precursorstransfected-MM cells. Effect of miRNA-15a and -16-1 on signaling cascades have been evaluated by western-blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding-enzymelinked-immunosorbent-assay-based-assay. Angiogenesis has been studied in vitro and in vivo using the chorioallantoic-membrane model. Results: We identified a MM-specific miRNA signature characterized by decreased expression of miRNA-15a, -16-1 and increased expression of miRNA-222/-221/-382/-181a/-181b (P < .01). MM cell lines showed similar miRNA expression pattern to primary MM tumor cells. qRT-PCR was performed on matched samples and showed expression patterns similar to those observed in miRNA analysis. Using algorithms commonly used to predict human miRNA gene targets, predicted targets of decreased miRNAs in MM patients included pro-angiogenic cytokines/oncogenes/cell cycle regulators/ NFkB activators. Conversely, predicted target genes for increased miRNAs in MM included cell cycle inhibitors/suppressors of cytokine signaling/pro-apoptotic factors. Functional studies revealed that miRNA-15a and -16-1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/cdk6/pRb protein expression; phase-G1-cell-cycle arrest; as compared to either scramble probe-transfected or nontransfected-MM cells. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of phospho-IkB in the cytoplasm; and decreased translocation of p-p65 from the cytoplasm to the nucleus. We also confirmed antiangiogenic properties of miRNA-15a and -16-1 both in vitro and in vivo. Conclusion: These data indicate that miRNAs play a pivotal role in the biology of MM; and provide the basis for the development of new miRNA-based targeted-therapies in this disease.”
B582 Unusual Monoclonal Gammopathies Detected in the Clinical Laboratory of a General Hospital CC Cellier,1 MK Sarda,1 J Troncy,2 CC Cellier1 1
Laboratoire Immunochimie Batiment 5, Hopital Edouard Herriot
Lyon, France; 2Hematologie Clinique, Hopital Edouard Herriot Lyon France
Introduction: Monoclonal gammopathies are a common finding in a laboratory of a general hospital. However, besides the usual monoclonal IgGs, IgMs, and IgAs, there are less common monoclonal components. Electrophoresis (EP) and electrophoresis-immunofixation (IFE) were reported by us and others as not sensitive enough to detect free light chains (FLC). Thus, the aim of this study is to report about the sensitivity of conventional techniques for screening and typing unusual M-components. Material and Methods: Routine screening
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for M-components was performed using capillary zone EP (Beckman), and ultra sensitive immunoelectrophoresis (IEP). Routine typing for M-components was performed using immunosubstraction performed with CZE, IFE with SPIFE 2000 (Helena). For some unusual Mcomponents typing was ascertained using immunoselection. Results: During a 7-year period of time (2001-2008) we have detected Mcomponents in the sera of 1859 patients. 37 patients presented with an unusual M-component (1,8%): 17 gamma heavy chain disease (GHCD), 13 IgD M-components, 4 alpha heavy chain disease (AHCD) proteins, 1 IgE M-component, 1 LC diclonal gammopathy and a monoclonal IgM lambda associated with a complete lack of kappa light chains. All IgD and IgE M-components were successfully screened with EP and typed with IFE. No AHCD protein could be screened with EP nor typed with IFE. One patient only had a typical immunoproliferative intestinal disease, the others having multiple myeloma, post transplantation non Hodgkin malignant lymphoma (NHML) and refractory anemia. Of the 17 GHCD proteins, 6 displayed no electrophoretic abnormalities and were exclusively characterized by an extra arch using IEP. Seven patients presented with lymphoproliferative syndrome, 3 with autoimmune diseases, 3 with NHML, 3 with MGUS and 1 with aids. Conventional typing techniques were unable to demonstrate the biclonality of the LC disease, while IEP and FLC quantisation did. Using IEP permitted to prove the lack of kappa chains in a sample with monoclonal IgM lambda. Conclusion: While conventional techniques screen and type successfully more than 98% of intact monoclonal immunoglobulins, there is still a need for other methods to detect either FLC or heavy chain disease proteins.
B587 A Novel Genetic Polymorphism in the DNA Polymerase Kappa Gene May Play a Role in Multiple Myeloma HR Morse, P Adamo, N Ladhani, L Zavridou, A McEwen University of the West of England
Introduction and Aims: DNA polymerase kappa (polk) is a “specialized” polymerase, which is able to insert the correct base opposite DNA lesions via the translesional synthesis pathway (TLS)1. Replication with polk of normal DNA is however inaccurate. Recent evidence demonstrates polk may be over-expressed in some tumors and thus suggests a role for polk in drug resistance and genome instability, due to its ability to “ignore” DNA lesions from chemotherapeutics and to create induce mutations when competing with replicative polymerases for the DNA template respectively. This study describes the identification of a novel genetic polymorphism in the polk gene promoter, which may influence gene expression. Since we previously observed high levels of polk expression in multiple myeloma (MM) samples, we wished to determine the frequency of the polymorphism in MM patients. Methods: The promoter (1Kb) of polk was examined by PCR-SSCP analysis, using overlapping primer pairs. Electrophoretic mobility shifts of amplicons were detected for one primer pair, suggestive of polymorphism. This proved to be a rare polymorphism, with 4 heterozygotes in 122
Clinical Lymphoma & Myeloma Supplement February 2009
XII International Myeloma Workshop individuals (freq = 0.016). DNA from wild-type and polymorphic individuals was cloned into pGEM T-easy vectors to separate the alleles for sequencing. Transfected colonies were analyzed by SSCP to identify the clones containing polymorphic alleles. DNA sequencing was performed on both native DNA and plasmids containing the wild type and polymorphic alleles. Results and Conclusions: Polymorphic sequences demonstrated a G to C transversion at position -517 according to the transcription start site 13. Promoter analysis suggested that no transcription factor binding motifs lie close to the polymorphism, although loss of a CpG methylation site may result, potentially increasing expression. DNA from MM patients demonstrated 4 heterozygotes in 45 individuals (frequency = 0.044) which was statistically significant (P < .0005). Increased polk expression in MM may contribute to the observed drug resistance and/or may create the complex karyotype observed. Future work is focusing on increasing the MM population size to confirm the frequency observed, correlating differential gene expression of polk with and without the polymorphism and eventually correlation with clinical progression and prognosis during DNA damaging therapies.
B589 Melphalan Produces an Osteoclast-Like Phenotype in MM Cell Lines and Exposes a MM “Stem Cell” HR Morse, G Newman, H Todman, D Martin, D Baker University of the West of England
Introduction: Osteolysis causes significant morbidity in MM patients. The underlying etiology is incompletely understood, but one possible mechanism is the unbalanced homeostasis of the RANK/RANKL/OPG pathway, where increased RANKL expression with down-regulation of the decoy receptor OPG increases osteoclast activity. MM cell lines cultured in the presence of Melphalan, demonstrated spontaneous morphological change, specifically the osteoclast-like appearance described by Calvani. This study describes these drug-induced changes. Methods: Myeloma cell lines (RPMI8226, U266, Jim1, LP1) and the B cell line AGLCL, were cultured with and without melphalan. Cells were maintained for 6-8 weeks. Cells were observed for morphology by microscopy and flow cytometry, and for phenotypic changes by flow cytometry (CD44) and immunofluoresent localisation of RANKL. Results and Conclusion: Melphalan exposure induced large, granular, multinucleated adherent cells in RPMI8226, LP1 and Jim1, but not AGLCL or U266. Flow cytometry confirmed the increased size and granularity. Initial treatment created adherent cells, but prolonged treatment cycles reduced adherence and cells were observed as lymphocytic-type cells of reduced size and granularity. These cells were resistant to further melphalan toxicity and we termed these “myeloma stem cells”, since cultures were reconstituted from these cells. After 2-3 weeks recovery, re-treatment with melphalan achieved re-adherence and a similar cycle of activity, but complete culture depletion was not achieved by continuous melphalan exposure. The adhesion molecule CD44 did not appear to influence adherence as melphalan led to inconsistent expression on the cell surface. CD44
was down-regulated in RPMI cells, however further treatments, regained CD44 expression and exceeded that of untreated cells. U266 cells however, demonstrated up-regulation of CD44. RANKL expression was localized cytoplasmically in all non-adherent cells, but was cell membrane localised in all adherent multinucleated cells, offering an explanation of bone loss in MM. These initial data demonstrate MM cells can respond morphologically and phenotypically to melphalan exposure, possibly into osteoclast-like cells. Current studies are further characterising these adherent cells to confirm their similarity to those observed by Calvani. Additionally, we intend to further characterise the myeloma “stem cell” and confirm it as such.
B590 Myeloma Cells as Measles Virus Delivery Vehicles SJ Russell, C Liu, HT Ong, KW Peng Department of Molecular Medicine, Mayo Clinic
Introduction and Aims: Attenuated measles viruses administered by the intravenous route mediate regression of myeloma xenografts in SCID mice. MV-NIS, an engineered measles virus encoding the thyroidal sodium iodide symporter (NIS) is undergoing phase I clinical evaluation (intravenous administration) in patients with myeloma at Mayo Clinic. Intravenously administered measles viruses can be neutralized by circulating antiviral antibodies or sequestered in liver and spleen. They also extravasate poorly from tumor neovessels so only a small fraction reaches the target site. Cellular carriers might overcome these delivery barriers by shielding the virus from antibodies and transporting it to its target site. Mature plasma cells migrate efficiently from lymph nodes to bone marrow. Myeloma cells retain this bone marrow tropism and may therefore be ideal carriers. For safety reasons, if myeloma cells were to be used as virus delivery vehicles, they would have to be lethally irradiated before administering them to patients. Materials and Methods: Biodistribution of luciferase-expressing MM1 myeloma cells infected with a recombinant measles virus encoding red fluorescent protein (MV-RFP) was determined after intravenous injection in SCID mice bearing orthotopic disseminated KAS6/1 human myeloma disease. Lethally irradiated MM1 cells were then evaluated for susceptibility to measles and ability to transfer infectivity to KAS6/1 myeloma cells by cell-cell heterofusion. Finally, SCID mice bearing disseminated KAS6/1 myeloma disease (6 r 109 cells intavenously 30 days prior) were given intravenous lethally irradiated, MV-NIS infected MM1 cells (106 cells per injection, 40 Gy irradiation, 8 mice per group) and followed for signs of disease progression. Results and Conclusions: Abundant MV-RFP infected MM1 cells were identified in bone marrow extracted from femurs of KAS 6/1 tumor-bearing mice three days after cell administration. Lethally irradiated MM1 cells remained susceptible to measles infection and could subsequently transfer infectivity to viable KAS6/1 cells by heterofusion. Mice treated intravenously with lethally irradiated, MV-NIS infected MM1 cells showed significant slowing of tumor progression and prolongation of survival compared to untreated mice. Irradiated myeloma cells deserve further invesigation as potentially ideal cellular vehicles for intravenous delivery of oncolytic measles virus to sites of myeloma growth.
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B582 Unusual Monoclonal Gammopathies Detected in the Clinical Laboratory of a General Hospital CC Cellier,1 MK Sarda,1 J Troncy,2 CC Cellier1 1
Laboratoire Immunochimie Batiment 5, Hopital Edouard Herriot
Lyon, France; 2Hematologie Clinique, Hopital Edouard Herriot Lyon France
Introduction: Monoclonal gammopathies are a common finding in a laboratory of a general hospital. However, besides the usual monoclonal IgGs, IgMs, and IgAs, there are less common monoclonal components. Electrophoresis (EP) and electrophoresis-immunofixation (IFE) were reported by us and others as not sensitive enough to detect free light chains (FLC). Thus, the aim of this study is to report about the sensitivity of conventional techniques for screening and typing unusual M-components. Material and Methods: Routine screening
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for M-components was performed using capillary zone EP (Beckman), and ultra sensitive immunoelectrophoresis (IEP). Routine typing for M-components was performed using immunosubstraction performed with CZE, IFE with SPIFE 2000 (Helena). For some unusual Mcomponents typing was ascertained using immunoselection. Results: During a 7-year period of time (2001-2008) we have detected Mcomponents in the sera of 1859 patients. 37 patients presented with an unusual M-component (1,8%): 17 gamma heavy chain disease (GHCD), 13 IgD M-components, 4 alpha heavy chain disease (AHCD) proteins, 1 IgE M-component, 1 LC diclonal gammopathy and a monoclonal IgM lambda associated with a complete lack of kappa light chains. All IgD and IgE M-components were successfully screened with EP and typed with IFE. No AHCD protein could be screened with EP nor typed with IFE. One patient only had a typical immunoproliferative intestinal disease, the others having multiple myeloma, post transplantation non Hodgkin malignant lymphoma (NHML) and refractory anemia. Of the 17 GHCD proteins, 6 displayed no electrophoretic abnormalities and were exclusively characterized by an extra arch using IEP. Seven patients presented with lymphoproliferative syndrome, 3 with autoimmune diseases, 3 with NHML, 3 with MGUS and 1 with aids. Conventional typing techniques were unable to demonstrate the biclonality of the LC disease, while IEP and FLC quantisation did. Using IEP permitted to prove the lack of kappa chains in a sample with monoclonal IgM lambda. Conclusion: While conventional techniques screen and type successfully more than 98% of intact monoclonal immunoglobulins, there is still a need for other methods to detect either FLC or heavy chain disease proteins.
B587 A Novel Genetic Polymorphism in the DNA Polymerase Kappa Gene May Play a Role in Multiple Myeloma HR Morse, P Adamo, N Ladhani, L Zavridou, A McEwen University of the West of England
Introduction and Aims: DNA polymerase kappa (polk) is a “specialized” polymerase, which is able to insert the correct base opposite DNA lesions via the translesional synthesis pathway (TLS)1. Replication with polk of normal DNA is however inaccurate. Recent evidence demonstrates polk may be over-expressed in some tumors and thus suggests a role for polk in drug resistance and genome instability, due to its ability to “ignore” DNA lesions from chemotherapeutics and to create induce mutations when competing with replicative polymerases for the DNA template respectively. This study describes the identification of a novel genetic polymorphism in the polk gene promoter, which may influence gene expression. Since we previously observed high levels of polk expression in multiple myeloma (MM) samples, we wished to determine the frequency of the polymorphism in MM patients. Methods: The promoter (1Kb) of polk was examined by PCR-SSCP analysis, using overlapping primer pairs. Electrophoretic mobility shifts of amplicons were detected for one primer pair, suggestive of polymorphism. This proved to be a rare polymorphism, with 4 heterozygotes in 122
Clinical Lymphoma & Myeloma Supplement February 2009
XII International Myeloma Workshop individuals (freq = 0.016). DNA from wild-type and polymorphic individuals was cloned into pGEM T-easy vectors to separate the alleles for sequencing. Transfected colonies were analyzed by SSCP to identify the clones containing polymorphic alleles. DNA sequencing was performed on both native DNA and plasmids containing the wild type and polymorphic alleles. Results and Conclusions: Polymorphic sequences demonstrated a G to C transversion at position -517 according to the transcription start site 13. Promoter analysis suggested that no transcription factor binding motifs lie close to the polymorphism, although loss of a CpG methylation site may result, potentially increasing expression. DNA from MM patients demonstrated 4 heterozygotes in 45 individuals (frequency = 0.044) which was statistically significant (P < .0005). Increased polk expression in MM may contribute to the observed drug resistance and/or may create the complex karyotype observed. Future work is focusing on increasing the MM population size to confirm the frequency observed, correlating differential gene expression of polk with and without the polymorphism and eventually correlation with clinical progression and prognosis during DNA damaging therapies.
B589 Melphalan Produces an Osteoclast-Like Phenotype in MM Cell Lines and Exposes a MM “Stem Cell” HR Morse, G Newman, H Todman, D Martin, D Baker University of the West of England
Introduction: Osteolysis causes significant morbidity in MM patients. The underlying etiology is incompletely understood, but one possible mechanism is the unbalanced homeostasis of the RANK/RANKL/OPG pathway, where increased RANKL expression with down-regulation of the decoy receptor OPG increases osteoclast activity. MM cell lines cultured in the presence of Melphalan, demonstrated spontaneous morphological change, specifically the osteoclast-like appearance described by Calvani. This study describes these drug-induced changes. Methods: Myeloma cell lines (RPMI8226, U266, Jim1, LP1) and the B cell line AGLCL, were cultured with and without melphalan. Cells were maintained for 6-8 weeks. Cells were observed for morphology by microscopy and flow cytometry, and for phenotypic changes by flow cytometry (CD44) and immunofluoresent localisation of RANKL. Results and Conclusion: Melphalan exposure induced large, granular, multinucleated adherent cells in RPMI8226, LP1 and Jim1, but not AGLCL or U266. Flow cytometry confirmed the increased size and granularity. Initial treatment created adherent cells, but prolonged treatment cycles reduced adherence and cells were observed as lymphocytic-type cells of reduced size and granularity. These cells were resistant to further melphalan toxicity and we termed these “myeloma stem cells”, since cultures were reconstituted from these cells. After 2-3 weeks recovery, re-treatment with melphalan achieved re-adherence and a similar cycle of activity, but complete culture depletion was not achieved by continuous melphalan exposure. The adhesion molecule CD44 did not appear to influence adherence as melphalan led to inconsistent expression on the cell surface. CD44
was down-regulated in RPMI cells, however further treatments, regained CD44 expression and exceeded that of untreated cells. U266 cells however, demonstrated up-regulation of CD44. RANKL expression was localized cytoplasmically in all non-adherent cells, but was cell membrane localised in all adherent multinucleated cells, offering an explanation of bone loss in MM. These initial data demonstrate MM cells can respond morphologically and phenotypically to melphalan exposure, possibly into osteoclast-like cells. Current studies are further characterising these adherent cells to confirm their similarity to those observed by Calvani. Additionally, we intend to further characterise the myeloma “stem cell” and confirm it as such.
B590 Myeloma Cells as Measles Virus Delivery Vehicles SJ Russell, C Liu, HT Ong, KW Peng Department of Molecular Medicine, Mayo Clinic
Introduction and Aims: Attenuated measles viruses administered by the intravenous route mediate regression of myeloma xenografts in SCID mice. MV-NIS, an engineered measles virus encoding the thyroidal sodium iodide symporter (NIS) is undergoing phase I clinical evaluation (intravenous administration) in patients with myeloma at Mayo Clinic. Intravenously administered measles viruses can be neutralized by circulating antiviral antibodies or sequestered in liver and spleen. They also extravasate poorly from tumor neovessels so only a small fraction reaches the target site. Cellular carriers might overcome these delivery barriers by shielding the virus from antibodies and transporting it to its target site. Mature plasma cells migrate efficiently from lymph nodes to bone marrow. Myeloma cells retain this bone marrow tropism and may therefore be ideal carriers. For safety reasons, if myeloma cells were to be used as virus delivery vehicles, they would have to be lethally irradiated before administering them to patients. Materials and Methods: Biodistribution of luciferase-expressing MM1 myeloma cells infected with a recombinant measles virus encoding red fluorescent protein (MV-RFP) was determined after intravenous injection in SCID mice bearing orthotopic disseminated KAS6/1 human myeloma disease. Lethally irradiated MM1 cells were then evaluated for susceptibility to measles and ability to transfer infectivity to KAS6/1 myeloma cells by cell-cell heterofusion. Finally, SCID mice bearing disseminated KAS6/1 myeloma disease (6 r 109 cells intavenously 30 days prior) were given intravenous lethally irradiated, MV-NIS infected MM1 cells (106 cells per injection, 40 Gy irradiation, 8 mice per group) and followed for signs of disease progression. Results and Conclusions: Abundant MV-RFP infected MM1 cells were identified in bone marrow extracted from femurs of KAS 6/1 tumor-bearing mice three days after cell administration. Lethally irradiated MM1 cells remained susceptible to measles infection and could subsequently transfer infectivity to viable KAS6/1 cells by heterofusion. Mice treated intravenously with lethally irradiated, MV-NIS infected MM1 cells showed significant slowing of tumor progression and prolongation of survival compared to untreated mice. Irradiated myeloma cells deserve further invesigation as potentially ideal cellular vehicles for intravenous delivery of oncolytic measles virus to sites of myeloma growth.
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