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Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells showed profound expansion of clonal cells in both un-switched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper and effector and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. This study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies. This article is protected by copyright. All rights reserved.

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As4S4/ZnS/Fe3O4 (1:4:1), As4S4/ZnS/Fe3O4 with folic acid (FA), and As4S4/ZnS/Fe3O4 with FA and albumin with reduced survival MM cell lines and primary MM samples by each of 3NP. Cytotoxic effects of 3NPs were associated with caspase- and mitochondria-dependent apoptosis induction and reduced c-Myc expression. Modulation of cell cycle regulators, such as p-ATM/ATM and p-ATR/ATR, and increases in p-Chk2, cyclin B1, and histones were accompanied by G2/M arrest triggered by 3NPs. In addition, 3NPs activated several myeloma-related signaling, including JNK1/2/3, ERK1/2 and mTOR. To overcome BM microenvironment-mediated drug resistance, nanocomposites retained its anti-MM activity in the presence of stroma. 3NPs significantly decreased the stem cell-like side population in MM cells, even in the context of stroma. We observed strong synergistic effects of 3NPs combined with lenalidomide, pomalidomide, or melphalan, suggesting the potential of these combinations for future clinical studies.

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As

The aim of our prospective, interventional, pre-post, single arm study was to supplement the lack of knowledge of the effect of short-term Pilates intervention on selected blood parameters of healthy women. Female volunteers were recruited for 2-weeks Pilates intervention. Blood has been collected and anthropometric parameters were measured before and after exercise period (EP). Plasma insulin, cortisol, and dehydroepiandrosterone sulphate levels, erythrocyte antioxidant activity, glutathione levels, NK cytotoxicity and plasma cytokines were analysed. We found a decrease in erythrocyte antioxidant enzymes SOD and GPx activity; GSH levels; in the pro-inflammatory chemokine MCP-1 and trend to reduction in MIP-1β, PDGF and VEGF levels in plasma. NK cell cytotoxic activity increased after Pilates EP in the percentage of specific lysis at 25:1 effector: target (E:T) ratio and the same trend was observed at all E:T ratios as well as in the amount of lytic units per 107 cells. Our findings show that Pilates exercise may improve NK cell immune response and inflammatory milieu in plasma of healthy women.

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As4S4) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G2/M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumors due to the absence of biomarkers for early-stage detection and poor response to therapy. Since mounting evidence supports the role of microbiota composition in tumorigenesis and cancer treatment, the link between microbiome and PDAC has been described. In this review, we summarize the current knowledge regarding the impact of the gut and oral microbiome on the risk of PDAC development. Microenvironment-driven therapy and immune system interactions are also discussed. More importantly, we provide an overview of the clinical trials evaluating the microbiota role in the risk, prognosis, and treatment of patients suffering from PDAC and solid tumors. According to the research findings, immune tolerance might result from the microbiota-derived remodeling of pancreatic tumor microenvironment. Thus, microbiome profiling and targeting represent the potential trend to enhance antitumor immunity and improve the efficacy of PDAC treatment.

The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms.

Multiple myeloma (MM), the second most common hematologic malignancy worldwide, is a B cell malignancy characterized by high frequency of intra-clonal diversity within malignant plasma cells (PC) in the bone marrow (BM). To better understand the myeloma heterogeneity within its complex pathophysiology, we performed large-scale data-driven mass cytometry (CyTOF) analysis in cohort of 188 bone marrow (BM) samples from multiple myeloma (MM) patients compared to 10 age-matched healthy donors (HD). Our design focused on profiling of PC intra and inter-neoplastic heterogeneity based on molecular perturbations of transcriptional factors and signaling regulators and stemness-controlling markers ensuring development of B cell lymphopoiesis within myelomagenesis encompassing the different clinical spectra of pre-malignant/asymptomatic (16 MGUS and 25 SMM) and active symptomatic stages (43 NDMM and 104 relapsed or relapsed/refractory MM patients) of MM pathogenesis. Moreover, interaction of PC disease status with the immune ecosystem of myeloma microenvironment was evaluated as well. To distinguish tumor-driven specific immune changes from myeloma immune ecosystem, we observed that cell frequency of cytotoxic naïve and effector cells, g/dT, and early monocytes, myelocytes and erythroblasts immune subsets was significantly reduced in both premalignant and active MM stages. In contrast, mostly innate immune clusters including non-canonical monocytes, myeloblasts, and mature neutrophils, erythroblasts and platelets were present at a higher frequency across all MM stages versus HD. To evaluate cell distribution of B lymphopoiesis in MM disease stages, switched memory B cells and plasmablasts clusters were upregulated in premalignant stage MGUS compared to HD. Similar observations were detected in SMM and NDMM versus HD, with the highest abundance of PC clusters in NDMM. The downregulation of cell distribution in B cell progenies, immature and transitional B cells, and un-switched memory B cell clusters was observed in NDMM and relapsed/refractory MM patients. Furthermore, MM patients treated with Revlimid-Velcade-Dexamethasone therapy had decrease frequency of specific PC clusters and un-switched and transitional B cell clusters. In addition, our data revealed immunophenotyping aberrancies present not only in PC clusters but also across all myeloma B lymphomagenesis in BM samples from MM patients. In-depth characterization of malignant plasma cells, significant variations were detected in PC clusters of MM cohort based on different expression of IRF4, c-Myc, CD28, CD117, and FGFR-3, however with homogenous expression of sXBP1, and MMSET which differ in all 4 MM stages compared to HD. Significant upregulation of CD47 was showed in all PC clusters of MM cohort. Moreover, PC clusters differ in intra-clonal expression of self-renewing/stemness markers CD184, Notch-1, Oct3/4, KLF-4, Sox-2, and Nanog, supporting the idea of sub-clonal variations insight of MM tumor. This study might provide the rational for prediction of MM patient status and design of targeted therapy in MM on personalized bases. This work was supported by REA grant agreement No. 609427-SASPRO 0064/01/02, TRS-2015-00000170, APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Jamroziak:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: 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, Research Funding; Bristol-Myers Squibb: Research Funding. Kastritis:Prothena: Honoraria; Genesis: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: 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, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

In this study, arsenic sulphide As4S4nanoparticles have been prepared, by high-energy wet milling, in the presence of sodium dodecylsulphate, which acts a surfactant. Solid state properties of the nanoparticles were characterised by XRD, Raman scattering, specific surface area and particle size distribution. Changes in surface areas of the particles, in the 0.2 - 5.4 m2g-1range, and nanosize distributions, in the 100 - 250 nm range, characterise the surface and morphological properties of nanorealgar. Raman scattering revealed various species in the milled sample that indicate a disproportionate reaction (3As4S4→ 4As2S3+ 4As) occurring as a consequence of milling. Anticancer effects, of the milled species, were confirmed for the human multiple myeloma U266 and OPM1 cell lines. Dissolution experiments in simulated gastric fluid show a possibility for the application of the realgar nanoparticles as an oral dose in future arsenic drug cancer treatments.

In this study, the nanosized arsenic sulphide As4S4 particles have been prepared by mechanical activation in a planetary mill. The bulk and surface properties of the milled particles were characterized by XRD and XPS methods as well as by surface area and particle size distribution measurements. For assessment of arsenic sulphide biological activity, the viability tests of multiple myeloma cancer cells have been applied. The obtained results show the arsenic sulphide nanoparticles properly milled and modified by mechanical activation can be valuated as a potential drug for cancer treatment.

In the past two decades nanocrystalline sulphide semiconductors have attracted considerable interest due to their intriguing properties and structure diversities. Arsenic show interesting solid state phenomena as well as therapeutic effects for various diseases. In this study, the nanosized realgar As 4 S 4 particles (d – 144 nm) have been prepared by a high-energy milling. The solid state properties of the nanoparticles milled under various experimental conditions were characterized by XRD method, by measurement of specific surface area and particle size distribution in nanosized region. In biological tests the sensitivity of human cancer cell lines for the realgar nanoparticles has been clearly demonstrated.

To address a precise view into molecular mechanisms of apoptotic signaling pathways after single- or combinatory treatments with specific NF-κB- or proteasome inhibitors and/or cisplatin (CDDP), flow cytometry and western blotting of the cell proteome in human ovarian chemosensitive- and CDDP-resistant cell lines were used. We report here that proteasome inhibition (but not NF-κB inhibition) caused marked alterations in the cell proliferation and cell cycle, as well as in the levels of signaling anti- and pro-apoptotic proteins PARP, NF-κB, IκB-α, Bcl-2, Bax, and lysosome-associated LAMP-1 and ATP-7B molecules in particular proteome fractions. These findings refer to the possibility of regulation of CDDP resistance, inclusive the capacity of lysosomes to export CDDP in certain human ovarian cancer cells by proteasome inhibition.

Metastasis as a complex process involves loss of adhesion, migration, invasion and proliferation of cancer cells. Sulforaphane (SFN) is one of naturally occurring cancer chemopreventive isothiocyanates found in cruciferous vegetables, consumption of which has been associated with reduced risk of cancer. In this study, we describe effect of SFN on various aspects determining invasive behavior of MDA-MB-231 human breast carcinoma cells. We studied modulation of molecules associated with epithelial to mesenchymal transition (EMT), hypoxic marker CA IX and mitochondrially located peripheral benzodiazepine receptor (PBR) using flow cytometry, gene expression of matrix metalloproteinases MMP1, 3, 7, 9, 14, transcription factors POU5F1 and Twist1 mRNA by RT PCR, and cytokine production by multiplex bead assay. SFN downregulated PBR and vimentin expression in a dose dependent manner, but significantly affected neither HIF-1alpha, nor CA IX protein expression, nor VEGF and GLUT1 mRNA levels. Among studied MMPs, MMP7 and MMP14 mRNA were downregulated while no apparent effect on MMP1, MMP3 and MMP9 was observed. Further, we found significant down regulation of Twist1 and POU5F1, transcription factors that mediate EMT and the self-renewal of undifferentiated embryonic stem cells. SFN reduced also the production of pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha, IFN-gamma, immunomodulating cytokine IL-4 and growth factors involved in angiogenesis PDGF and VEGF. Our study shows that SFN efficacy is associated with the reversal of several biological characteristics connected with EMT or implicated in the matrix degradation and extracellular proteolysis, as well as with reduced production of pro-inflammatory cytokines and pro-angiogenic growth factors in MDA-MB-231 cells.

BioBran, enzymatically modified arabinoxylan from rice bran was tested for its possible effects on in vitro maturation of human dendritic cells (DC). Immature DC (iDC) derived from plastic-adhered, IL-4 and GM-CSF treated peripheral monocytes (Mo) were further cultured with cytokine maturation mix 1 (CMM1; TNF-α, IL-1β and IL-6) or CMM2 (LPS and IFN-γ) to induce their maturation into mature DC (matDC1 or matDC2, respectively). Different concentrations of BioBran (10, 100, 400 and 1000 μg/ml) were applied in the presence or absence of relevant CMM to assess the effects of BioBran on DC maturation processes. BioBran induced maturation of iDC, as these cells cultured with IL-4/GM-CSF/BioBran down-regulated CD14 and CD1a antigens on cell surface and significantly increased expression of maturation marker CD83. The increase of surface density of costimulatory molecules CD80 and CD86 on iDC in the presence of BioBran was also observed. In addition, BioBran induced functional maturation of iDC, confirmed by decreased endocytic activity of iDC. Furtheremore, BioBran enhanced maturation potential of cytokine mixes, as both matDC1 and matDC2 exposed to BioBran completely lost CD14 and upregulated CD83, CD80 and CD86 antigens, in comparison to DC matured with the relevant CMM alone. BioBran also increased CD123 antigen expression on all DC subsets. Interestingly, matDC2 matured in the presence of BioBran (400μg/ml) expressed higher levels of CD123 and lower levels of CD11c cell surface antigens, the phenotype represented by CD11c dim CD123 bright plasmacytoid DC population. These data demonstrate that BioBran is a potent enhancer of DC maturation and suggest that BioBran might be a useful agent to create the environment that favours DC maturation.

Intra-clonal heterogeneity in malignant plasma (PC) cells and clonal B cells has been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity together with humoral immunity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. By a large cohort of MM (N = 100) and WM (N = 50) patient samples, we studied evolution of bone marrow patient samples from premalignant stages (MGUS and smoldering MM) to malignant stages (MM and WM) versus age-matched healthy donors (HD) by combining immunophenotypic, molecular, and cytogenetic approaches. We examined phenotypic and molecular features of intra-clonal architecture of malignant PC in MM and clonal B cells in WM together with humoral immunity on a single cell level using mass cytometry (CyTOF). CyTOF is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers can simultaneously assess immunophenotyping and analysis of signaling in single cell. To investigate phenotypic profile and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses revealed that clonal PC clustered separately from B cells with high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARα2. sXBP-1 was significantly upregulated in all MM stages. Furthermore, expression of stem cell markers Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Both MM and WM are characterized by immune dysfunction; therefore we used CyTOF technology to define the complex immune profile in MM and WM patient samples. Therefore, we examined changes in B cell development, T cell subsets, NK and NKT cells as well as monocytic, granulocytic, erythroid and platelet lineages during evolution of both diseases vs. HD on phenotypic and molecular level. For example, plasmacytoid dendritic cells (pDC) were increased in MM with significantly decreased PD-1 expression on pDC in MM compared to MGUS (P = 0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) in MM. A better understanding of inter- and intra-clonal heterogeneity together with host immunity on the phenotypic and molecular/signaling level in MM and WM will provide the framework for identifying and validating novel personalized targeted therapies. Citation Format: Jana Jakubikova, Danka Cholujova, Teru Hideshima, Jacob Laubach, Nikhil C. Munshi, Steven P. Treon, Paul G. Richardson, Efstathios Kastritis, David M. Dorfman, Kenneth C. Anderson. Phenotypic and molecular characterization of inter- and intraclonal heterogeneity in multiple myeloma and Waldenstrom macroglobulinemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2004. doi:10.1158/1538-7445.AM2015-2004

Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples. Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software. Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. Disclosures No relevant conflicts of interest to declare.

Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software. Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented. Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.

Introduction Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy. Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation. Methods and Results First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment. To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment. Conclusions Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Introduction The neoplastic bone marrow (BM) milieu or so-called myeloma niche plays an essential role in initiation, development, and progression of multiple myeloma (MM). It is known that regulatory signaling molecules and cell-cell crosstalk interactions involved in the normal hematopoietic stem cell niche have also been implicated in MM. Defining phenotypic features and molecular signatures of the neoplastic BM niche in MM will provide the framework for future development of new treatment strategies targeting MM cells and their BM niche. Methods and Results To investigate the neoplastic BM microenvironment of MM patients, we compared a special gel-based 3-dimensional (3-D) model with a 2-dimensional (2-D) system for culturing MM patient-derived mesenchymal stem cells (MM-MSCs) ex-vivo. In the gel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections between clusters to create a MM niche-like structure. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of MSC-specific markers including CD73, CD90, CD105, CD166, and HLA-ABC. On the other hand, 3-D MM-MSCs clusters lacked expression of CD45, CD34 and HLA-DR. In order to confirm differentiation potential of 3-D MM-MSC, we cultured them with differentiation media towards either osteogenic or adipogenic lineages. The lineage differentiation capacity of the 3-D system was comparable to the 2-D MM-MSCs, with stronger calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. Moreover, the production of osteopontin and angiopoietin-2 was evident in 3-D MM-MSCs. In these models of the neoplastic BM microenvironment in MM, significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p To define the functional myeloma niche, key hematopoietic and/or neoplastic niche molecules were investigated by analyzing 30 MM-MSCs and 5 healthy volunteers MSCs. MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin by flow cytometry analysis, reflecting the MM BM niche. Higher pattern of cytokine production of leptin, SCF and sTie-2 was evident in 3-D MM-MSCs compared to 2-D system, as well as cancer biomarkes like IGFBP-1, sEGFR and sHER2neu performed by multiplex luminex technology. Next, we observed statistically significant higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p To investigate the pathogenesis of MM in neoplastic BM niche, we co-cultured BM aspirates of MM cells (N=30) with MM-MSCs in vitro. Co-culturing BM aspirates with (autologous) MM-MSCs increased MM cells (CD38+/CD138+/CD19-) as well as stimulated their proliferation (determined by CFSE proliferation dye) in 3-D vs. 2-D system. MM cells in 3-D MM-MSCs were 4-fold more resistant to bortezomib and pomalidomide as well as melphalan, compared to 2-D. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features, were significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Conclusions Our 3-D MM-MSCs model mimics the neoplastic BM microenvironment and myeloma niche in vitro, and may be useful to identify novel targets, validate targeted therapies and determine mechanisms of drug resistance in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Abstract 1812 The neoplastic bone marrow (BM) milieu plays important roles in the development of multiple myeloma (MM). Specifically, cell-cell crosstalk interactions and regulatory signaling molecules involved in the normal hematopoietic stem cell niche have also been implicated in initiation, development, and progression of MM. Moreover, this “myeloma niche” serves as a sanctuary site for MM stem cells. The increasing knowledge of interactions between complex of MM cells and other normal hematopoietic cells and also non-hematopoietic mesenchymal stem cells and their progeny may enhance our understanding of MM cell biology and pathogenesis as well as provide the basis for novel therapeutic strategies. In order to develop an experimental system mimicking the specialized properties of the neoplastic BM microenvironment, we used hydrogel based 3-dimensional (3-D) versus 2-dimensional (2-D) models together with MM-derived mesenchymal stem cells (MM-MSCs) to create MM niche-like structures in vitro. In this 3-D model, MM-MSCs grew with the matrix fibers and at day 7 formed stable compact clusters, with fibrous meshwork-like structure among them. Furthermore, we showed tri-lineage differentiation capacity of 3-D comparable to 2D MM-MSCs towards osteogenic, adipogenic, and chondrogenic lineages. Our 3-D MM-MSCs model also showed calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. The phenotypic profile of MM-MSCs grown in the 2-D vs. 3-D model using MSC-specific markers (such as CD166, HLA-ABC, CD73, CD105, and CD90) did not reveal any significant changes, however CD271 was more highly expressed in the 3-D model, while CD146 was overexpressed in 2-D cultures. Because the stem cell niche is enriched with extracellular matrix (ECM) molecules resulting in an overexpansion of the stem cell pool, we studied whether expression of these molecules was conserved in the 3-D model. Analyzing 30 MM-MSCs vs. 5 normal donor MSCs, we observed that the 3-D MM-MSCs expressed significantly high level of ECM molecules including laminin, fibronectin, collagen I, collagen IV and vitronectin (p To mimic the functional marrow niche of MM in our model, we cultured the tumor cells from MM patients (N=30) with their autologous MM-MSCs in vitro. MM plasma cells (identified as CD38/CD138+ cells) were increased in percentage and proliferation after 14 days in the 3-D vs. 2-D model. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features (Jakubikova J et al., Blood 2011) were also significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Our study shows that hydrogel based 3-D MM-MSCs model forms a functional niche in vitro, and may be useful to identify novel targets and validate therapies directed at MM stem cells in the MM BM niche. Disclosures: Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Oncopep: Patents & Royalties. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees.

Background Study results demonstrated that IFN augments BEV activity and improves median PFS in pts with mRCC. Thus, combination BEV + IFN is a standard first-line treatment option for mRCC. Combining BEV with the mTOR inhibitor EVE may be an efficacious and well-tolerated treatment option. The open-label, phase II RECORD-2 trial compared first-line EVE + BEV and IFN + BEV in mRCC. Patients and methods: Therapy-naive pts with clear cell mRCC and prior nephrectomy were randomized 1:1 to BEV 10 mg/kg IV every 2 weeks with either EVE 10 mg oral daily or IFN (9 MIU SC 3 times/week, if tolerated). Tumour assessments were every 12 weeks. Primary objective was treatment effect on progression-free survival (PFS) per central review based on an estimate of the chance of a subsequent phase III trial success (50% threshold for phase II success). Results In EVE + BEV (n = 182) and IFN + BEV (n = 183) arms, median age was 60/60 years, 76/72% of pts were men, MSKCC risk was favourable/intermediate/poor in 36/57/7% and 36/57/7% of pts, and 43/46% of pts had >2 organs involved, respectively. For EVE + BEV and IFN + BEV, median treatment duration was 8.5/8.3 months, respectively; 23/26% of pts discontinued due to AEs. In EVE + BEV and IFN + BEV arms, median PFS by central review was 9.3/10.0 months (HRIFN/EVE, 0.91; 95% CI, 0.69-1.19; P =0.485), respectively; probability of subsequent phase III success was 5.1%. Results of central and local PFS analysis were consistent. Objective response rate was 27/28% in EVE + BEV and IFN + BEV arms, respectively. Median overall survival (OS) was not reached in the EVE + BEV arm and was 25.9 months (95% CI: 21.1, 30.2) in the IFN + BEV arm. Most frequent AEs (%) were stomatitis (63), proteinuria (49), diarrhoea (39), hypertension (38), and epistaxis (35) in EVE + BEV arm and decreased appetite (45), fatigue (41), proteinuria (37), and pyrexia (35) in IFN + BEV arm. Conclusions In RECORD-2, PFS and tolerability were similar for first-line EVE + BEV and IFN + BEV. Final OS analysis will occur after 2-year follow-up. Disclosure A. Ravaud: Alain Ravaud is a member of global, European, and/or French boards on urological tumors for Pfizer, Novartis, GlaxoSmithKline, Bayer-Schering, and Dendreon, and has received institutional grant support from Pfizer, Novartis, and Roche. O. Anak: Ozlem Anak is an employee of Novartis Pharma AG. D. Pelov: Diana Pelov is an employee of Novartis Pharmaceuticals Corporation. A. Louveau: Anne-Laure Louveau is an employee of Novartis Pharma S.A.S. T. M-H: Tay M-H is a speaker for an advisory board for Novartis Pharmaceuticals Corporation. B. Melichar: Bohuslav Melichar has received honoraria from Novartis and Roche and served on an advisory board for Roche. All other authors have declared no conflicts of interest.

Background Testicular germ-cell tumours (TGCTs) represent a model for the cure of cancer. Nonetheless, a small proportion of patients develop disease recurrence. We investigated cytokines and angiogenic factors (CAFs) in patients with TGCTs. We aimed to link the CAF profile to PFS and select candidate predictive and prognostic markers for further study. Methods In this ongoing translational study, plasma from 55 patients with TGCTs was collected. The concentrations of 51 plasma CAFs were measured pretreatment (n = 47) and on day 22 (n = 30) using multiplex bead arrays (Human Group I and II cytokine panels and TGF beta by Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA). We investigated the association between baseline levels of CAFs and clinico-pathological variables. Results We observed elevated level of transforming growths factor beta1 (TGF-b1), IL-2Ra, CXCL9, CXCL10, IFN-gama, macrophage inflammatory protein-1b (MIP-1b), and platelet-derived growth factor-BB (PDGF-BB) in seminoma patients compared to non-seminoma. Patients with poor prognosis according to IGCCCG have elevated pretreatment level of beta-nerve growth factor (NGF-b) and stromal cell-derived factor-1 (SDF-1a) compared to patients with good/intermediate prognosis. Patients with metastatic disease had elevated pretreatment level of stem cell growth factor beta (SCGF-b) and PDGF-BB compared to patients with stage I disease. Conclusions Using CAF profiling, we revealed differences in subgroups of TGCTs patients. We suggest that this platform may provide valuable insights into TGCTs biology, and could be useful in identification of new therapeutic targets. Disclosure All authors have declared no conflicts of interest.

Abstract 954 Introduction: Multiple myeloma (MM), a malignancy of plasma cells, remains incurable even though newly established regimens incorporating conventional and novel agents are capable of achieving complete clinical and biochemical remissions in a sizeable subset of MM patients. This indicates the presence of a distinctive drug-resistant sub-population of MM cells, which might be responsible for tumor re-growth. Methods and Results: To evaluate this phenomenon, we performed flow cytometry-based Hoechst 33342 staining assay to evaluate the existence and characteristics in MM of a population known as side population (SP), which has been identified in diverse neoplasias and has been reported to exhibit “stem-like” features. We specifically performed flow cytometry or ImageStream analyses to quantify the SP population in a panel of 18 MM cell lines. These analyses revealed heterogeneity in the proportion of SP fractions in different cell lines and indicated the lack of correlation between CD138 expression and SP fraction. We observed that, after sorting of the SP and non-SP fractions, SP cells have the potential to generate colonies in CFU assays, and regenerate a population resembling the original, pre-sorted, one. Moreover, SP cells had a high proliferation index compared to non-SP cells. We also observed that most MM cells lines express significantly higher levels of ABCG2 transcripts in their sorted SP fraction compared to their non-SP cells. The mRNA profile results were further corroborated by functional assays, where SP cells showed more pronounced efflux of the known ABCG2 substrate mitoxantrone, consistent with the association of SP phenotype with ABCG2 expression. Interestingly, at concentrations and durations of drug exposure that did not substantially affect the viability of non-SP cells, the immunomodulatory thalidomide derivative (IMiDs) lenalidomide significantly decreased the percentage and clonogenicity of SP cells. This response was accompanied by a distinct pattern of changes in diverse signaling pathways in SP cells (including phosphorylation changes in Akt, GSK-3a/b, MEK1, c-Jun, p53, p38 MAPK, p70S6K and STAT3) and increase of CD138-/low+ phenotype. Because the BM microenvironment is considered a key regulator of MM cell biological behavior, we evaluated the impact of bone marrow stromal cells (BMSCs) on the behavior of SP cells and observed that BMSCs led to increased percentage, viability, as well as proliferation potential of SP cells. Interestingly, IMiDs abrogated this stimulatory effect of stromal cells by significantly decreasing SP cell percentages. In these co-culture models, we identified several cytokines, including IL-1b, IL-10, IL-17; chemokines (MIP-1a, MIP-1b and IP-10) and growth factors such as GM-CSF and VEGF, which were up-regulated in co-culture MM cells with stromal cells compared to stroma alone or MM cells alone. Significant modulation in the production of these cytokines and chemokines was detected after IMiD treatment of MM cells only, as well as in co-culture model. Conclusion: Identifying the clonogenic population of SP cells could facilitate the development of new strategies targeting subpopulations of MM cells with drug resistant properties. Our study raises the intriguing possibility that IMiDs could represent such a strategy that can target SP cells and counteract their resistance to different conventional, and perhaps some novel, therapies. In addition, our data suggest that further studies of this tumor cell population are warranted towards to the goal of developing new strategies to treat MM. Disclosures: Laubach: Novartis: Consultancy, Honoraria. Richardson:Millenium: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding.