Comparison of Newly Diagnosed and Relapsed Refractory Multiple Myeloma Using Transcriptional Profiling of Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1555-1555
Author(s):  
Shaji Kumar ◽  
Philip R. Greipp ◽  
Jessica L. Haug ◽  
Michael Kline ◽  
Wee Joo Chng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Differential Expression ◽  
Median Survival ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Expression Profiles ◽  
Significant Proportion ◽  
Newly Diagnosed ◽  
Myeloma Cells

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that is incurable with current approaches. The median survival for patients with MM is around four years and a significant proportion of patients experience a course characterized by multiple relapses treated with different therapies. The median survival for patients relapsing after the initial therapy is nearly 18 months and successive treatment strategies result in decreasing response durations, likely reflecting acquired drug resistance. In order to better understand the biological changes associated with advanced, relapsed, refractory MM, we compared gene expression profiles (GEP) of malignant plasma cells isolated from patients with relapsed refractory MM and compared them to plasma cells from patients with newly diagnosed MM. Methods: In order to obtain two relatively homogenous group of patients, we compared samples from 44 patient with newly diagnosed MM enrolled in the ECOG E1A00 clinical trial (comparing thalidomide and dexamethasone to dexamethasone alone) to 44 patients with relapsed refractory MM enrolled in a phase II trial of Velcade (SUMMIT), where most patients had four or more previous relapses. Plasma cells from bone marrow aspirates were separated by magnetic bead selection of CD138 positive cells and studied using Affymetrix HG-U133A chips using standard methodology. The arrays were analyzed using Genespring 7.2 software following GCRMA normalization and genes with differential expression between the two datasets were examined. Differentially expressed genes were further analyzed using Ingenuity Pathways Analysis program. Results: A total of 864 genes were identified which were at least two fold and significantly different between the newly diagnosed and relapsed patients. Using Ingenuity software, 437 of these genes were mapped to different biological networks. Examination of the canonical pathways demonstrated several important cellular pathways differentially regulated between the two groups. Several important mediators of the cytokines, receptors and respective signaling pathways appear to be down regulated in the relapsed group and included IGF-1, HGF, SDF-1 alpha, gp130 and importantly the MEK/ERK pathway. Additionally expression of adhesion molecules such as VCAM1 and PECAM was decreased in the relapsed group compared to newly diagnosed pts. There appear to increased tissue hypoxia in the relapsed marrow as indicated by up regulation of HIF-1 alpha as well increased levels of Placental growth factor. Myeloma cells from relapsed disease were characterized by decreased expression of mcl1, FLIP1, and bcl-xL and increased caspase 8 relative to newly diagnosed group. Also seen was decreased expression of the glucocorticoid and interferon receptors in the relapsed setting. Conclusion: Comparison of the GEP between MM cells from newly diagnosed and relapsed pts demonstrates important differences that have potential biological relevance. The plasma cell in the relapsed setting appears to be more independent of the tumor microenvironment. Additionally, differential expression of some of the genes provides clues to mechanisms of drug resistance commonly observed in the relapsed pts. We are in the process of validating some of the key findings from these analyses.

scholarly journals Peripheral blood monoclonal plasma cells as a predictor of survival in patients with multiple myeloma [see comments]

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1780-1787 ◽  
Author(s):  
TE Witzig ◽  
MA Gertz ◽  
JA Lust ◽  
RA Kyle ◽  
WM O'Fallon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Prognostic Factors ◽  
High Risk ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Labelling ◽  
Newly Diagnosed ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Beta 2 Microglobulin

Abstract The purpose of this study was to quantitate the number and labeling index of monoclonal plasma cells in the blood of patients with newly diagnosed multiple myeloma (MM) to learn if these values were independent prognostic factors for survival. Patients were candidates for this study if they had untreated myeloma requiring therapy, were evaluated at our institution between 1984 and 1993, and had a sample of blood analyzed with a sensitive immunofluorescence technique for monoclonal plasma cells and the blood B-cell labelling index (BLI). The % blood monoclonal plasma cells (%BPC) and the BLI were analyzed along with stage, marrow plasma cell LI, % marrow plasma cells, calcium, creatinine, albumin, beta-2-microglobulin, and C-reactive protein as univariate and multivariate factors for survival. Eighty percent of the 254 patients accrued to this study had monoclonal BPC detected. The median % BPC was 6% and 57% (144 of 254) of patients had a high number (> or = 4%). Patients with > or = 4% BPC had a median survival of 2.4 years vs 4.4 years for those with < 4% BPC (P < .001). The BLI was also prognostic (P = .008). In a multivariate analysis, the % BPC, age, albumin, stage, marrow plasma cell LI, and the BLI were independent factors for survival. The %BPC and the marrow plasma cell LI best separated the group into low, intermediate, and high risk myeloma with median survivals of 52, 35, and 26 months, respectively. Patients with high %BPC were less likely to have lytic bone disease from their MM (P = .002). The %BPC and the BLI are independent prognostic factors for survival and are useful in identifying patients as low, intermediate, and high risk. Clonal cells in the blood should be quantified in future clinical trials for myeloma.

High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3820-3820
Author(s):  
Yi Tao ◽  
Zhimin Gu ◽  
Ye Yang ◽  
Hongwei Xu ◽  
Xiaojing Hu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Caspase 9 ◽  
Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

Targeting the Serine-Threonine Kinase PIM2: Implications in IL-6 Mediated Survival in Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3401-3401
Author(s):  
Jayakumar R Nair ◽  
Tyger L Howell ◽  
Justin Caserta ◽  
Carmen M Baldino ◽  
Gerald Fetterly ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Death ◽  
Plasma Cells ◽  
Serine Threonine Kinase ◽  
Myeloma Cells ◽  
Threonine Kinase ◽  
Equity Ownership ◽  
Microarray Expression

Abstract Despite major advances in chemotherapy, multiple myeloma remains incurable and in need of new therapies that target novel pathways. Insufficient understanding of the molecular pathways that regulate survival in myeloma is a major impediment towards designing better therapies to prolong survival in patients or even cure the disease. This necessitates the identification of new protein targets that are crucial for the growth and survival of multiple myeloma. Just like normal plasma cells, MM cells also depend on their interactions with bone marrow stromal cells (BMSC) for survival and production of essential growth factors. We have previously shown that MM cells interact with dendritic cells (DC) in the microenvironment and in vitro can stimulate DC to produce IL-6 (ASH2010#132, ASH2011 #147, ASH2012#722). Our recent publications show that when MM cells are not in direct contact with DC, the IL-6 produced by DC can protect MM cells against dexamethasone induced cell death, while neutralizing the IL-6 with antibodies can reverse that effect (Nair et al., 2011). Unfortunately, exactly how this survival response is mediated in MM is not very clear. PIM2, a serine threonine kinase, part of the proto-oncogene group of PIM kinases has been implicated in survival in several types of cancers including prostate cancer and multiple myeloma. In our lab, microarray gene expression analysis of publicly available datasets (Figure 1) show a trend towards increased expression of PIM2 in plasma cells from myeloma patients (left panel), and significantly in the poor prognosis subgroup MAF (Zhan et al., 2006) (right panel). For the first time we show that IL-6 produced by DC may be protecting myeloma cells by up regulating PIM2 and inactivating a major protein translation inhibitor 4EBP1, which also happens to be a PIM2 target. We show that silencing PIM2 with siRNA down regulates PIM2 activity and reverses the inactivation of 4EBP1, while the latter is known to cause cell death in myeloma. We also demonstrate that neutralizing IL-6 in MM cells that either don’t produce IL-6 on their own (MM.1S) or those that do (U266), abrogates extraneous DC-IL6 ability to induce PIM2 and its downstream target 4EBP1. Recombinant IL-6 also provided similar induction of PIM2 in myeloma and increased 4EBP1 phosphorylation, which was again reversed by neutralizing the antibody against IL-6. In myeloma patients, the use of dexamethasone in frontline therapies is often complicated by the ability of the bone marrow environment to produce IL-6 that not only induce increased proliferation of MM but also help resist dexamethasone mediated cell death in myeloma. Interestingly, when we used a novel PIM2 inhibitor, JP_11646 (kindly provided by Jasco Pharmaceuticals, LLC), it not only arrested IL-6 induced proliferation even at sub-lethal doses, but also prevented IL-6 mediated rescue of myeloma cells (Figure 2). This suggests that PIM2 might be a major player in IL-6 mediated drug resistance in myeloma and targeting it may help to subvert IL-6 mediated survival in myeloma. Through RT-PCR and westerns, we also show that IL-6 modulates PIM2 expression and activity resulting in increased 4EBP1 phosphorylation (Figure 3). This was abrogated when PIM2 activity was inhibited by JP_11646 (Figure 3). We also present data that suggests IL-6 via PIM2 may be regulating other anti-apoptotic molecules downstream of IL-6 receptors including MCL-1, that is vital to MM survival. Developing PIM2 targeted therapies provides an exciting opportunity to affect the myeloma tumor microenvironment where MM induced IL-6 production from BM could be inducing drug resistance. Figure 1: Microarray expression ofPIM2 in myeloma and MAF Figure 1:. Microarray expression ofPIM2 in myeloma and MAF Figure 2: PIM2 inhibition abrogates IL-6 induced MM proliferation (A) and protection (B). Figure 2:. PIM2 inhibition abrogates IL-6 induced MM proliferation (A) and protection (B). Figure 3: Inhibiting PIM2 activity prevents PIM2 induced phosphorylation of 4EBP1 by IL-6 in myeloma Figure 3:. Inhibiting PIM2 activity prevents PIM2 induced phosphorylation of 4EBP1 by IL-6 in myeloma Disclosures Caserta: Jasco Pharmaceuticals LLC: Equity Ownership. Baldino:Jasco Pharmaceuticals LLC: Equity Ownership.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

scholarly journals Multiple Myeloma Macrophages: Pivotal Players in the Tumor Microenvironment

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Simona Berardi ◽  
Roberto Ria ◽  
Antonia Reale ◽  
Annunziata De Luisi ◽  
Ivana Catacchio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Microenvironment ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Proteolytic Enzymes ◽  
Vasculogenic Mimicry ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Growth And Survival

Tumor microenvironment is essential for multiple myeloma (MM) growth, progression, and drug resistance through provision of survival signals and secretion of growth and proangiogenic factors. This paper examines the importance of macrophages within MM bone marrow (BM) microenvironment, referred to as MM-associated macrophages, as a potential niche component that supports tumor plasma cells. These macrophages are derived from peripheral blood monocytes recruited into the tumor. Upon activation by MM plasma cells and mesenchymal stromal cells, macrophages can release growth factors, proteolytic enzymes, cytokines, and inflammatory mediators that promote plasma cell growth and survival. Macrophages promote tumor progression through several mechanisms including angiogenesis, growth, and drug resistance. Indeed, these macrophages are essential for the induction of an angiogenic response through vasculogenic mimicry, and this ability proceeds in step with progression of the plasma cell tumors. Data suggest that macrophages play an important role in the biology and survival of patients with MM, and they may be a target for the MM antivascular management.

scholarly journals The expression of p53 protein in patients with multiple myeloma

2007 ◽  
Vol 135 (1-2) ◽  
pp. 43-47 ◽  
Author(s):  
Olivera Markovic ◽  
Dragomir Marisavljevic ◽  
Vesna Cemerikic ◽  
Maja Perunicic ◽  
Milica Colovic
Keyword(s):  
Multiple Myeloma ◽  
Clinical Significance ◽  
Median Survival ◽  
Plasma Cells ◽  
Histological Grade ◽  
Stage I ◽  
Newly Diagnosed ◽  
P53 Expression ◽  
P53 Immunoreactivity ◽  
Histological Stage

Introduction: Although mutations of p53 are one of the most often acquired genetic changes in malignant tumors, these mutations are rare events in patients with newly diagnosed multiple myeloma (MM). Moreover, there are a few literature data about clinical significance of p53 overexpression in multiple myeloma. Objective The aim of our study was to evaluate the clinical significance of p53 immunoexpression in multiple myeloma. Method A total of 58 patients with newly diagnosed MM (26 females and 32 males, mean age 62 years) were enrolled in the study. The diagnosis of MM was made according to criteria of Chronic Leukemia-Myeloma Task Force. Clinical staging was done according to Durie and Salmon classification (4 patients had disease stage I, 15 patients stage II and 39 patients stage III). The histological grade and histological stage were determined according to predominant plasma cell morphology and volume of myeloma infiltration, respectively. Standard immunohistochemical analysis with p53 antibody in B5-fixed and paraffin- embedded bone marrow specimens was used to evaluate the expression of p53 in myeloma cells. The specimens were considered positive when ?5% of plasma cells exhibited clear nuclear positivity. Results Out of 58 patients, p53 expression was detected in 9 (15.52%). No significant correlation was found between p53 expression and clinical stage (I+II vs. III), ?2-microglobulin level (?6 mg/L vs. >6mg/L), histological grade (I vs. II+III), histological stage (<20% vs. 21-50% vs. >50%) and the extent of osteolytic lesions (?3 vs. >3 lesions). Median survival of patients with p53 immunoreactivity in =>5% of plasma cells was 10 months, whilst median survival of patients with p53 immunoreactivity in <5% of plasma cells was 36 months. However, such difference was not significant (p=0.2). Conclusion The frequency of p53 immunoexpression in our group of newly diagnosed MM was relatively low. Although p53 immunoexpression was not associated with clinical and histological features of more aggressive disease, or with shorter survival, further investigations of larger group of patients will lead to final conclusions.

A Trangenic Mouse Model of Plasma Cell Malignancy Shows Cytogenetic and Gene Expression Profiles Similar to Human Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 619-619
Author(s):  
Kristin Boylan ◽  
Mary A. Kvitrud ◽  
Brian G. Van Ness
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Chromosomal Abnormalities ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Rt Pcr ◽  
Mouse Tumors ◽  
Human Multiple Myeloma

Abstract Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. Human plasma cell tumors are genetically diverse, with no single chromosomal abnormality defining the disease, however, dysregulation of the genes c-myc and bcl-xl are both commonly observed. We have previously shown that targeted expression of c-myc and bcl-xl transgenes in mouse plasma cells produces malignancy which displays features of human myeloma such as localization of tumor cells to the bone marrow and lytic bone lesions. Tumors are also present at extramedullary sites (Cheung et al., J. Clin. Invest.113: 1763, 2004). Tumors rapidly develop (median 16 weeks) in 100% of mice, and can be adoptively transferred to syngeneic controls using as few as 1 million tumor cells to produce tumors in as few as 10 days. Adoptive transfer of similar cell numbers from younger double transgenic mice, without evidence of malignancy, results in increased tumor latency (&gt;8 weeks) or the absence of tumor formation, suggesting that an accumulation of genetic changes is required for tumor development. In order to understand the specific genetic alterations required for tumor progression and for localization of tumors to the bone marrow vs extramedullary sites, we have undertaken a detailed analysis of plasma cell tumors in myc/bcl-xl mice and have begun to compare them with human multiple myeloma. Analysis of cell surface markers shows the majority of tumors have a plasmablast phenotype, expressing CD138+, B220+, CD38+, and CD19+. This result is confirmed by RT-PCR for B cell and plasma cell specific markers Pax5, Xbp1 and Blimp1, which can be detected in tumor samples. In addition, transcripts for Mip1α, EZH2, and Dusp6, genes shown to be upregulated in human myeloma, can also be detected in the mouse myc/bcl-xl tumors. Spectral karyotype analysis of metaphase chromosomes from primary tumor cell cultures demonstrates that a variety of chromosomal abnormalities are present in mouse tumors, including trisomies and translocations, similar to what is observed in human myeloma. The most frequently aberrant chromosomes are 12 and 16, followed by chromosomes 1 and 4. Interestingly, two common sites for translocations were identified; 12F which corresponds to the mouse immunoglobulin heavy chain locus, and 4D, which corresponds to a genomic region containing genes for plasma cell tumor susceptibility (Bliskovsky et al., PNAS100:14982, 2003). Further characterization of these translocations are being done to identify the precise breakpoints involved, and analysis of gene expression by RT-PCR and microarray analysis will be correlated to specific chromosomal abnormalities. Additionally, global gene expression profiles from myc/bcl-xl tumor cell cultures have been compared to existing profiles of human myeloma (Zhan et al., Blood99: 1745, 2002). Our preliminary comparison of gene expression profiles from myc/bcl-xl tumors to human myeloma tumors with high myc expression show the mouse tumors are more similar to human tumors than to normal plasma cells. These data suggest the myc/bcl-xl mouse tumors are similar to a subset of human myelomas, and will provide insight into the specific genes and pathways underlying human disease.

Angiogenesis in Multiple Myeloma (MM): Angiogenic Switch or Reflexion of Plasma Cell Number? A Gene Expression Based Survey in Primary Myeloma Cells and the Bone Marrow Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3405-3405
Author(s):  
Dirk Hose ◽  
John DeVos ◽  
Christiane Heiß ◽  
Jean-Francois Rossi ◽  
Angela Rösen-Wolff ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Empirical Bayes ◽  
Plasma Cells ◽  
De Novo ◽  
Cell Number ◽  
Bone Marrow Microenvironment ◽  
Myeloma Cells ◽  
Angiogenic Switch

Abstract BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma. However, two etiologic hypotheses have been proposed: an angiogenic switch (i.e. differential or de novo expression of pro/antiangiogenic genes in MM), and, alternatively an effect of increased plasma cell number. AIM of this study was to investigate the angiogenic signature of multiple myeloma cells (MMC), normal bone marrow plasma cells (BMPC), the bone marrow microenvironment (BMME) and cellular subfractions therein. PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG) / 63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. Whole bone marrow (n=49) and FACSAria sorted subfractions thereof (n=5) were investigated. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. p-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). HGF expression was verified by real time RT-PCR and western blotting. Based on Medline review, we established a list of 89 pro- and 56 antiangiogenic genes and investigated their expression according to the stage of disease: BMPC vs. MGUS, SD stage I (asymptomatic myeloma) vs. SD stage II/III (symptomatic myeloma requiring therapy). RESULTS. BMPC express pro- (e.g. VEGFA) and antiangiogenic genes (e.g. TIMP2). Only one pro-angiogenic gene (hepatocyte growth factor, HGF) is significantly overexpressed in MMC compared to BMPC. HGF has previously been linked with myeloma progression and induction of angiogenesis. Six antiangiogenic genes (TIMP2, SERPINF1, COL18A1, PF4, THBS1, CXCL14) are downregulated in MMC compared with BMPC. Compared to healthy donors, the BMME of MM shows a significant downregulation of PLAU (urokinase, antiangiogenic) and upregulation of TNF(proangiogenic). CONCLUSION. Upregulation of HGF-expression, downregulation of TIMP2, SERPINF1, COLA18A1, PF4, THBS1 and CXCL14 expression in MMC as well as downregulation of PLAU and upregulation of TNFα in the BMME seem to indicate an “angiogenic switch”. However, given the relatively low number of differentially expressed genes (7/145) and the expression of angiogenic genes by BMPC, an effect caused by an increasing number of plasma cells might be evenly important.

A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1907-1907
Author(s):  
Eva Sahakian ◽  
Jason B. Brayer ◽  
John Powers ◽  
Mark Meads ◽  
Allison Distler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

CXCR3 Binding Chemokines MIG, IP-10 and ITAC Are Predictors of Overall Survival in Newly Diagnosed Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2052-2052
Author(s):  
Arnold Bolomsky ◽  
Niklas Zojer ◽  
Martin Schreder ◽  
Heinz Ludwig
Keyword(s):  
Multiple Myeloma ◽  
Overall Survival ◽  
Plasma Cells ◽  
In Silico Analysis ◽  
Serum Levels ◽  
Newly Diagnosed ◽  
Myeloma Cells ◽  
Detection Call ◽  
Silico Analysis

Abstract Background. The chemokine receptor CXCR3 and its binding molecules MIG, IP-10 and ITAC have been associated with tumor progression, immune escape and angiogenesis in several human malignancies. In multiple myeloma (MM), CXCR3 binding molecules were shown to induce migration of MM cells without effecting proliferation. More recent results suggest a tumor suppressive activity of IP-10. Presently, information about the precise role of CXCR3 binding chemokines in MM is limited and evidence for their clinical significance is lacking. Therefore we aimed to evaluate the prognostic relevance of CXCR3 binding chemokines in patients with MM. Patients and Methods. Serum levels of MIG, IP-10 and ITAC were analyzed by FACS-CBA array in 65 newly diagnosed MM patients. Expression of CXCR3 and its binding molecules was also analyzed by quantitative PCR in 7 human MM cell lines (HMCLs) and in a publically available gene expression dataset (GSE2658). Further analysis of MIG serum levels was performed by ELISA in an extended cohort of MM (n=105) and MGUS patients (n=17), and in healthy volunteers (n=37). Results. Determination of serum levels by FACS-CBA revealed significant expression of MIG (range: 33.4 – 157 960 pg/ml) and IP-10 (12 - 4418.8 pg/ml), while ITAC (0 - 351.5 pg/ml) was only detectable in a subset (20 of 65) of patients. Interestingly, serum levels of all three molecules showed a positive correlation with each other (MIG vs. IP-10, R=0.38, P=0.002; MIG vs. ITAC, R=0.62, P<0.0001; ITAC vs. IP-10, R=0.41, P=0.0007). We also observed a significant correlation with beta 2 microglobulin (B2M) (MIG: R=0.45, P<0.0001; IP-10: R=0.36, P=0.003; ITAC: R=0.3, P=0.016) and a trend regarding ISS stage (MIG: R=0.23, P=0.06; IP-10: R=0.24, P=0.05; ITAC: R=0.11, P=0.39). Importantly, a significant association with overall survival (OS) was observed as well. Survival was significantly worse in patients with high compared to low MIG (median OS 25.3 months vs. not reached, P=0.003) and IP-10 (19.97 months vs. not reached, P=0.0006) as well as in patients with detectable compared to absent ITAC serum levels (19.97 vs. 65.8 months, P=0.019). In multivariate analysis, MIG (P=0.03) and ITAC (P=0.013) along LDH and calcium were revealed as independent predictors of survival. Expression of CXCR3 binding chemokines was rarely detected in HMCLs (1 of 7 expressed MIG, 3 of 7 IP-10 and 2 of 7 ITAC, respectively). In line with this, in-silico analysis of previously published primary MM cell samples (n=414) (GSE2658), showed a present detection call of MIG, IP-10 and ITAC in 51 (12.3%), 11 (2.7%) and 0 (0%) patients, respectively. In contrast, all three cytokines were detectable in 100% of bone marrow plasma cells of healthy donors, MGUS and smoldering MM patients in this dataset. Hence, CXCR3 binding chemokines are silenced in myeloma cells indicating that the increased serum levels of CXCR3 binding chemokines are derived from other cell types. As MIG serum concentration was identified as one of the most important predictors for OS, we studied the prognostic relevance of this molecule in an extended cohort (n=105) of MM patients by ELISA. Median MIG levels (161.3 pg/ml, range: 9.4-1966) were significantly elevated in newly diagnosed MM patients compared to MGUS (92.7 pg/ml, range: 6.29-1303.1) and healthy volunteers (106.2, range: 51–390.6 pg/ml). MIG levels were significantly correlated with B2M, ISS stage, calcium, albumin, LDH, hemoglobin and with age (R=0.466, P<0.001). Importantly, high MIG levels predicted adverse survival (17.0 months vs. not reached, P<0.001), which was upheld when age-adjusted cut-off levels were used. In accordance with our findings, in-silico analysis of MIG expression in purified plasma cells of MM patients (n=559) treated within the total therapy 2 and 3 protocol (GSE2658) revealed shorter OS in patients with a present compared to those with an absent detection call for MIG (P=0.004). Conclusion. Our findings depict MIG, IP-10 and ITAC as novel prognostic markers for shorter survival in newly diagnosed MM patients. High serum levels of CXCR3 binding chemokines in conjunction with silenced expression in MM cells may shield myeloma cells from immune attack as previously shown for T cell lymphomas. Further experiments will aim to confirm these initial results by extending our patient cohort and define the source as well as functional role of CXCR3 chemokines in MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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